TW201350506A - ST2 antigen binding proteins - Google Patents

Abstract

Described herein are compositions and methods related to antigen binding proteins that bind to human ST2, including antibodies. In particular embodiments, the disclosure provides fully human anti-ST2 antibodies and deriviatives and variants thereof. Further provided are nucleic acids encoding such antibodies and antibody fragments, variants, and derivatives. Also, provided are methods of making and using such antibodies including methods of treating and preventing autoimmune and inflammatory disorders.

Description

ST2 antigen binding protein

ST2 is a binding receptor for interleukin-33 (IL-33), an interleukin-33-dependent interleukin associated with IL-1 and IL-18 and is also known as NF-HEV or IL-1F11. ST2 appears to be a soluble non-signaling variant (soluble ST2 or sST2) that mediates cellular responses to IL-33 and both full-length transmembrane forms (FL ST2, ST2 or ST2L). The latter form is manifested on a wide range of cell types associated with pathological inflammation in a variety of disease settings. These include lymphocytes, especially T helper cells that express IL-5 and IL-13, natural killer (NK) cells, and natural killer-T (NKT) cells, as well as many so-called innate immune cells, such as mast cells, alkaloids. Sexual balls, eosinophils, macrophages, and congenital helper cells (also known as nuocytes ( Neill, Wong et al. 2010 )). Similar to IL-1 and IL-18, binding of IL-33 to ST2 on these cells results in the recruitment of co-receptors known as IL-1R helper proteins (AcP) and the activation of inflammatory signaling. Thus, IL-33 is capable of directly activating ST2 expressing cells or enhancing its activation when other activation stimuli are present. Examples of IL-33-induced cellular responses include the production of inflammatory interleukins such as IL-5, IL-6, IL-13, TNF, IFN-γ, and GM-CSF, as well as the production of chemotactic hormones such as CXCL8, CCL17. And CCL24. IL-33 has also been shown to enhance acute allergic responses by augmenting mast cell and basophil activation triggered by IgE receptor signaling or other mast cells and basophilic ball activators. IL-33 also enhances the recruitment, survival and adhesion properties of ST2-expressing immune cells and is therefore important in stimulating and maintaining cell inflammation in local tissues.

The proinflammatory effect of IL-33 on innate and adaptive immune cells ultimately promotes many pathological processes. In the lungs, these include increased airway inflammation, mucus production, airway hyperreactivity, and fibrosis remodeling. IL-33 can also cause local inflammation of the joints and high damage to the skin and joints by promoting the production of inflammatory mediators ( Verri, Guerrero et al. 2008; Xu, Jiang et al. 2008 ). Excess IL-33 has been associated with pathological collagen deposition and fibrosis and it also causes epithelial damage in the context of inflammatory bowel disease. IL-33 can also trigger anaphylactic shock ( Pushparaj, Tay et al. 2009 ) and can play an important role in allergic diseases via effective effects on basophilic globules and IgE-sensitized mast cells. Many of these diseases are chronic and progressively worse and difficult to treat and require more effective treatment.

Consistent with the biological effects recorded, there is several evidence that the IL-33/ST2 pathway causes human disease. For example, IL-33 is abnormally high in performance in diseases involving mucosal tissue inflammation and joint inflammation. These include asthma ( Prefontaine, Lajoie-Kadoch et al. 2009 ; Prefontaine, Nadiger et al. 2010 ), inflammatory bowel disease ( Belan, Nunez et al. 2010 ; Pastorelli, Garg et al. 2010 ; Sponheim, Pollheimer et al. 2010 ) and Rheumatoid arthritis ( Paler, Talabot-Ayer et al. 2009 ; Matsuyama, Okazaki et al. 2010 ). IL-33 is expressed in the skin of patients with psoriasis ( Theoharides, Zhang et al. 2010 ) and patients with atopic dermatitis ( Pushparaj, Tay et al. 2009 ) and also in the pathological environment of fibrosis (eg systemic sclerosis ( Yanaba) , Yoshizaki et al. 2011 ) ( Manetti, Ibba-Manneschi et al. 2009 ) and liver fibrosis ( Marvie, Lisbonne et al. 2009 )). The concentration of circulating soluble ST2 is also elevated in many disease states, further indicating the association between this interleukin pathway and such diseases. Examples include asthma ( Kuroiwa, Arai et al. 2001 ; Oshikawa, Kuroiwa et al. 2001; Ali, Zhang et al. 2009 ), chronic obstructive pulmonary disease ( Higerer, Lambers et al. 2009 ), pulmonary fibrosis ( Tajima, Oshikawa et al. 2003 ). , sepsis and trauma ( Brunner, Krenn et al. 2004 ), HIV infection ( Miyagaki, Sugaya et al. 2011 ), systemic lupus erythematosus ( Mok, Huang et al. 2010 ), inflammatory bowel disease ( Belan, Nunez et al. 2010 ) and Rheumatoid arthritis, sclerosis, Wegener's granulomatosis and Behchet disease ( Kuroiwa, Arai et al. 2001 ) and cardiovascular disease ( Shah and Januzzi 2010 ). IL-33 may cause eosinophilic inflammation and there is evidence that this pathway is involved in eosinophilic leukocyte-related diseases such as sinusitis and nasal polyps ( Plager, Kahl et al. 2010 ) and eosinophilic bronchitis ( Oshikawa) , Kuroiwa et al. 2001 ).

Genetic studies provide additional evidence linking the IL-33/ST2 pathway to human disease, which has identified IL-33 and/or ST2 genes that are significantly associated with increased disease risk or disease severity parameters in the general population. State. Several large genome-wide related studies have linked genetic variation in ST2 (IL1RL1) or IL-33 to increased risk of asthma ( Gudbjartsson, Bjornsdottir et al. 2009 ; Moffatt, Gut et al. 2010 ; Wu, Romieu et al. 2010 ) and other studies. This pathway has been genetically associated with increased asthma severity ( Al, Zhang et al. 2009 ) and collateral hyperactivity ( Reijmerink, Postma et al. 2008 ). Similar findings have been genetically shown to be associated with allergic conditions such as atopic dermatitis ( Shimizu, Matsuda et al. 2005 ), sinusitis ( Sakashita, Yoshimoto et al. 2008 ; Castano R 2009 ) and nasal polyps ( Buysschaert) , Grulois et al. 2010 ) related.

In summary, the association of this interleukin axis with several human diseases and its ability to promote many forms of harmful inflammation suggests that this line can be used as a target for therapeutic intervention.

The present invention provides anti-ST2 antigen binding proteins, such as antibodies and functional fragments thereof, having properties suitable for commercial production and human therapeutic applications. Anti-ST2 antigen binding proteins are particularly useful in methods of treating diseases and conditions associated with the IL-33/ST2 axis. Provided herein are ST2-binding antibodies that bind ST2 with high affinity and effectively block IL-33-binding thereby reducing IL-33 mediated signaling in cells.

In the first aspect, the ST2 antigen binding protein comprises a) and SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID The amino acid sequence described in NO: 104, SEQ ID NO: 105, SEQ ID NO: 163, SEQ ID NO: 164 or SEQ ID NO: 165 has at least 90% identity, at least 95% identity or consistent lightness a chain variable domain; b) with SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 The amino acid sequence set forth in SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 145, SEQ ID NO: 146 or SEQ ID NO: 147 has a heavy chain variable domain of at least 90% identity, at least 95% identity or identity; or c) a light chain variable domain of a) and a heavy chain variable domain of b).

Preferred antigen binding proteins of the first aspect include those comprising: a light chain variable structure having at least 90% identity, at least 95% identity or agreement with the amino acid sequence set forth in SEQ ID NO: 95 a domain and a heavy chain variable domain having at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 29; these include: and SEQ ID NO: 96 The amino acid sequence has at least 90% identity, at least 95% identity or identity of the light chain variable domain and is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 30, at least 95 % identical or identical heavy chain variable domains; these include: a light chain that is at least 90% identical, at least 95% identical or identical to the amino acid sequence set forth in SEQ ID NO: 97 A variable domain and a heavy chain variable domain having at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 31; these include: SEQ ID NO: The amino acid sequence of 98 having at least 90% identity, at least 95% identity or consistent light chain variable domain and The amino acid sequence set forth in SEQ ID NO: 32 has a heavy chain variable domain of at least 90% identity, at least 95% identity or identity; these include: the amine described in SEQ ID NO: 99 The acid sequence has at least 90% identity, at least 95% identity or one The light chain variable domain and the heavy chain variable domain having at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 33; these include: a light chain variable domain having at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 100 and having at least the amino acid sequence set forth in SEQ ID NO: 34 90% identity, at least 95% identity or consensus heavy chain variable domains; they comprise: at least 90% identity, at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 101 a light chain variable domain of the sex or consensus and a heavy chain variable domain having at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 35; A light chain variable domain having at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 102 and the amino acid sequence set forth in SEQ ID NO: 36 Heavy chain variable domains having at least 90% identity, at least 95% identity, or agreement; these include: SEQ ID NO: 10 The amino acid sequence of 3 having at least 90% identity, at least 95% identity or identity of the light chain variable domain and at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 37, At least 95% identical or identical heavy chain variable domains; these include: at least 90% identity, at least 95% identity or consistency with the amino acid sequence set forth in SEQ ID NO: 104 A chain variable domain and a heavy chain variable domain having at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 38; these include: SEQ ID The amino acid sequence of NO: 105 having at least 90% identity, at least 95% identity or identity of the light chain variable domain and at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 39 a heavy chain variable domain that is at least 95% identical or identical; they comprise: at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 163 a light chain variable domain and at least 90% identical to at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 145 Or a consensus heavy chain variable domain; these include: at least 90% identity, at least 95% identity, or one with the amino acid sequence set forth in SEQ ID NO: 164 a light chain variable domain and a heavy chain variable domain having at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 146; and the inclusion thereof a light chain variable domain having at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 165 and having the amino acid sequence set forth in SEQ ID NO: 147 A heavy chain variable domain that is at least 90% identical, at least 95% identical or identical.

In a second aspect, the ST2 antigen binding protein comprises a) and SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100 , SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 163, SEQ ID NO: 164 or SEQ ID NO: 165 a light chain variable domain in which the amino acid sequence differs by no more than 10 or no more than 5 amino acid additions, deletions or substitutions; b) and SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31. SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, a heavy chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions or substitutions in the amino acid sequence of SEQ ID NO: 145, SEQ ID NO: 146 or SEQ ID NO: 147; Or c) the light chain variable domain of a) and the heavy chain variable domain of b).

Preferred antigen-binding proteins of the second aspect include those comprising: no more than 10 or no more than 5 amino acid additions, deletions or substitutions to the amino acid sequence described in SEQ ID NO: 95 a chain variable domain and a heavy chain variable domain that differs from the amino acid sequence set forth in SEQ ID NO: 29 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions; : a light chain variable domain that differs from the amino acid sequence set forth in SEQ ID NO: 96 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions and with the amine described in SEQ ID NO: a heavy chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions or substitutions; the group consisting of: The amino acid variable sequence of SEQ ID NO: 97 differs by no more than 10 or no more than 5 amino acid additions, deletions or substitutions and the amino acid described in SEQ ID NO: 31 A heavy chain variable domain having a sequence that differs by no more than 10 or no more than 5 amino acid additions, deletions or substitutions; these include: a difference of no more than 10 from the amino acid sequence set forth in SEQ ID NO:98 a light chain variable domain with or without more than 5 amino acid additions, deletions or substitutions and no more than 10 or no more than 5 amino acid additions to the amino acid sequence set forth in SEQ ID NO:32, A deleted or substituted heavy chain variable domain; these include: a light that differs from the amino acid sequence set forth in SEQ ID NO: 99 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions a chain variable domain and a heavy chain variable domain that differs from the amino acid sequence set forth in SEQ ID NO: 33 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions; : no more than 10 or no more than 5 amino acid additions, deletions or subtractions from the amino acid sequence described in SEQ ID NO: 100 a light chain variable domain and a heavy chain variable domain that differs from the amino acid sequence set forth in SEQ ID NO: 34 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions; The light chain variable domain differing from the amino acid sequence described in SEQ ID NO: 101 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions and as set forth in SEQ ID NO: 35 a heavy chain variable domain in which the amino acid sequence differs by no more than 10 or no more than 5 amino acid additions, deletions or substitutions; these include: the amino acid sequence described in SEQ ID NO: 102 a light chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions or substitutions and no more than 10 or no more than 5 amines compared to the amino acid sequence set forth in SEQ ID NO:36 a heavy chain variable domain with added, deleted or substituted base acids; these include: no more than 10 or no more than 5 amino acid additions, deletions to the amino acid sequence described in SEQ ID NO:103 Or a substituted light chain variable domain and differs from the amino acid sequence set forth in SEQ ID NO: 37 by no more than 10 or no more than 5 a heavy chain variable domain with an amino acid added, deleted or substituted; these include: no more than 10 or no more than 5 amino acid additions to the amino acid sequence set forth in SEQ ID NO: 104 Light, missing or replaced a chain variable domain and a heavy chain variable domain that differs from the amino acid sequence set forth in SEQ ID NO: 38 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions; : a light chain variable domain that differs from the amino acid sequence set forth in SEQ ID NO: 105 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions and with the amines set forth in SEQ ID NO: 39 a heavy chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions or substitutions; these include: the difference from the amino acid sequence described in SEQ ID NO: 163 a light chain variable domain with more than 10 or no more than 5 amino acid additions, deletions or substitutions and no more than 10 or no more than 5 amino acids differs from the amino acid sequence set forth in SEQ ID NO:145 Addition, deletion or substitution of heavy chain variable domains; these include: no more than 10 or no more than 5 amino acid additions, deletions or substitutions to the amino acid sequence described in SEQ ID NO: 164 Light chain variable domain and no more than 10 or no more than 5 amines compared to the amino acid sequence set forth in SEQ ID NO:146 a heavy chain variable domain with added, deleted or substituted base acids; and the inclusion thereof comprising: no more than 10 or no more than 5 amino acid additions to the amino acid sequence set forth in SEQ ID NO:165, A deleted or substituted light chain variable domain and a heavy chain variable domain that differs from the amino acid sequence set forth in SEQ ID NO: 147 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions.

In a third aspect, the ST2 antigen binding protein comprises a light chain variable domain comprising: a) an LCDR1 that differs from the LCDR1 sequence described in SEQ ID NO: 106 by no more than three amino acid additions, deletions or substitutions An LCDR2 that differs from the LCDR2 sequence described in SEQ ID NO: 117 by no more than three amino acid additions, deletions or substitutions; and no more than three amino acid additions to the LCDR3 sequence described in SEQ ID NO: 128, a missing or substituted LCDR3; b) an LCDR1 that differs from the LCDR1 sequence described in SEQ ID NO: 107 by no more than 3 amino acid additions, deletions or substitutions; and a difference of no more than 3 from the LCDR2 sequence described in SEQ ID NO: 118 LCDR2 with an amino acid added, deleted or substituted; and no more than 3 amino acid additions, deletions or substitutions to the LCDR3 sequence described in SEQ ID NO: 129 LCDR3; c) LCDR1 having no more than 3 amino acid additions, deletions or substitutions from the LCDR1 described in SEQ ID NO: 108; no more than 3 amine groups differing from the LCDR2 sequence described in SEQ ID NO: 119 Acid-added, deleted or substituted LCDR2; and LCDR3 which differs from the LCDR3 sequence described in SEQ ID NO: 130 by no more than 3 amino acid additions, deletions or substitutions; d) and the LCDR1 sequence described in SEQ ID NO: 109 LCDR1 having a difference of no more than 3 amino acid additions, deletions or substitutions; LCDR2 differing from the LCDR2 sequence described in SEQ ID NO: 120 by no more than 3 amino acid additions, deletions or substitutions; and SEQ ID NO: 131 The LCDR3 sequence differs by no more than 3 amino acid additions, deletions or substitutions of LCDR3; e) an LCDR1 which differs from the LCDR1 sequence described in SEQ ID NO: 110 by no more than 3 amino acid additions, deletions or substitutions; LCDR2 which differs from the LCDR2 sequence described in SEQ ID NO: 121 by no more than 3 amino acid additions, deletions or substitutions; and no more than 3 amino acid additions or deletions from the LCDR3 sequence described in SEQ ID NO: 132 Or substituted LCDR3; f) differs from the LCDR1 sequence described in SEQ ID NO: 111 by no more than 3 amines LCDR1 with addition, deletion or substitution of a base acid; LCDR2 which differs from the LCDR2 sequence described in SEQ ID NO: 122 by no more than 3 amino acid additions, deletions or substitutions; and the difference from the LCDR3 sequence described in SEQ ID NO: 133 LCDR3 with no more than 3 amino acid additions, deletions or substitutions; g) LCDR1 differing from the LCDR1 sequence described in SEQ ID NO: 112 by no more than 3 amino acid additions, deletions or substitutions; and SEQ ID NO: 123 The LCDR2 sequence differs by no more than 3 amino acid additions, deletions or substitutions of LCDR2; and LCDR3 which differs from the LCDR3 sequence described in SEQ ID NO: 134 by no more than 3 amino acid additions, deletions or substitutions; An LCDR1 that differs from the LCDR1 sequence described in SEQ ID NO: 113 by no more than three amino acid additions, deletions or substitutions; no more than three amino acid additions, deletions from the LCDR2 sequence described in SEQ ID NO: 124 Or substituted LCDR2; and LCDR3 which differs from the LCDR3 sequence described in SEQ ID NO: 135 by no more than 3 amino acid additions, deletions or substitutions; i) differs from the LCDR1 sequence described in SEQ ID NO: 114 by no more than 3 LCDR1 with amino acid addition, deletion or substitution; and LCDR2 sequence as described in SEQ ID NO: 125 Column phase LCDR2 with no more than 3 amino acid additions, deletions or substitutions; and LCDR3 which differs from the LCDR3 sequence described in SEQ ID NO: 136 by no more than 3 amino acid additions, deletions or substitutions; j) and SEQ ID NO The LCDR1 sequence of 115: the LCDR1 with no more than 3 amino acid additions, deletions or substitutions; the LCDR2 which differs from the LCDR2 sequence described in SEQ ID NO: 126 by no more than 3 amino acid additions, deletions or substitutions; And LCDR3 which differs from the LCDR3 sequence described in SEQ ID NO: 137 by no more than 3 amino acid additions, deletions or substitutions; k) differs from the LCDR1 sequence described in SEQ ID NO: 116 by no more than 3 amino acid additions , missing or substituted LCDR1; LCDR2 which differs from the LCDR2 sequence described in SEQ ID NO: 127 by no more than 3 amino acid additions, deletions or substitutions; and differs from the LCDR3 sequence described in SEQ ID NO: 138 by no more than 3 LCDR3 added, deleted or substituted with an amino acid; l) LCDR1 differing from the LCDR1 sequence described in SEQ ID NO: 166 by no more than 3 amino acid additions, deletions or substitutions; and as described in SEQ ID NO: 169 LCDR2 sequence differs by no more than 3 amino acid additions, deletions or substitutions of LCDR2; and SEQ ID NO The LCDR3 sequence described in 172 differs by no more than 3 amino acid additions, deletions or substitutions of LCDR3; m) differs from the LCDR1 sequence described in SEQ ID NO: 167 by no more than 3 amino acid additions, deletions or substitutions of LCDR1 An LCDR2 that differs from the LCDR2 sequence described in SEQ ID NO: 170 by no more than three amino acid additions, deletions or substitutions; and no more than three amino acid additions to the LCDR3 sequence described in SEQ ID NO: 173, Missing or substituted LCDR3; or n) LCDR1 differing from the LCDR1 sequence described in SEQ ID NO: 168 by no more than 3 amino acid additions, deletions or substitutions; no more than the LCDR2 sequence described in SEQ ID NO: 171 LCDR2 with 3 amino acid additions, deletions or substitutions; and LCDR3 which differs from the LCDR3 sequence described in SEQ ID NO: 174 by no more than 3 amino acid additions, deletions or substitutions; and heavy chain variable structures comprising the following Domain: o) HCDR1 differing from the HCDR1 sequence set forth in SEQ ID NO: 40 by no more than 3 amino acid additions, deletions or substitutions; no more than 3 amino acids differs from the HCDR2 sequence set forth in SEQ ID NO: 51 Addition, deletion or substitution of HCDR2; and difference from the HCDR3 sequence described in SEQ ID NO:62 More than 3 An amino acid added, deleted or substituted HCDR3; p) an HCDR1 differing from the HCDR1 sequence set forth in SEQ ID NO: 41 by no more than 3 amino acid additions, deletions or substitutions; and HCDR2 as described in SEQ ID NO: 52 HCDR2 having a sequence difference of no more than 3 amino acid additions, deletions or substitutions; and HCDR3 differing from the HCDR3 sequence set forth in SEQ ID NO: 63 by no more than 3 amino acid additions, deletions or substitutions; q) and SEQ ID The HCDR1 sequence described in NO: 42 differs by no more than 3 amino acid additions, deletions or substitutions of HCDR1; HCDR2 differs from the HCDR2 sequence described in SEQ ID NO: 53 by no more than 3 amino acid additions, deletions or substitutions. And HCDR3 which differs from the HCDR3 sequence described in SEQ ID NO: 64 by no more than 3 amino acid additions, deletions or substitutions; r) differs from the HCDR1 sequence described in SEQ ID NO: 43 by no more than 3 amino acids Addition, deletion or substitution of HCDR1; HCDR2 differing from the HCDR2 sequence described in SEQ ID NO: 54 by no more than 3 amino acid additions, deletions or substitutions; and no more than the HCDR3 sequence described in SEQ ID NO: 65 3 amino acid added, deleted or substituted HCDR3; s) and HCDR1 sequence as described in SEQ ID NO: 44 HCDR1 with no more than 3 amino acid additions, deletions or substitutions; HCDR2 differing from the HCDR2 sequence described in SEQ ID NO: 55 by no more than 3 amino acid additions, deletions or substitutions; and SEQ ID NO: 66 The HCDR3 sequence differs by no more than 3 amino acid additions, deletions or substitutions of HCDR3; t) an HCDR1 which differs from the HCDR1 sequence described in SEQ ID NO: 45 by no more than 3 amino acid additions, deletions or substitutions; HCDR2 differing from the HCDR2 sequence set forth in SEQ ID NO: 56 by no more than 3 amino acid additions, deletions or substitutions; and no more than 3 amino acid additions or deletions compared to the HCDR3 sequence set forth in SEQ ID NO:67 Or substituted HCDR3; u) HCDR1 differing from the HCDR1 sequence set forth in SEQ ID NO: 46 by no more than 3 amino acid additions, deletions or substitutions; no more than 3 HCDR2 sequences as described in SEQ ID NO: 57 An amino acid added, deleted or substituted HCDR2; and HCDR3 differing from the HCDR3 sequence set forth in SEQ ID NO: 68 by no more than 3 amino acid additions, deletions or substitutions; v) and SEQ ID NO: 47 HCDR1 sequences differ by no more than 3 amino acid additions, deletions or substitutions of HCDR1; The HCDR2 sequence described in SEQ ID NO: 58 differs by no more than 3 amino acid additions, deletions or substitutions of HCDR2; and differs from the HCDR3 sequence set forth in SEQ ID NO: 69 by no more than 3 amino acid additions, deletions or Substituted HCDR3; w) HCDR1 differing from the HCDR1 sequence set forth in SEQ ID NO: 48 by no more than 3 amino acid additions, deletions or substitutions; no more than 3 amines from the HCDR2 sequence set forth in SEQ ID NO:59 HCDR2 with addition, deletion or substitution of a base acid; and HCDR3 which differs from the HCDR3 sequence described in SEQ ID NO: 70 by no more than 3 amino acid additions, deletions or substitutions; x) and HCDR1 as described in SEQ ID NO: 49 HCDR1 having a sequence difference of no more than 3 amino acid additions, deletions or substitutions; HCDR2 differing from the HCDR2 sequence set forth in SEQ ID NO: 60 by no more than 3 amino acid additions, deletions or substitutions; and SEQ ID NO: The HCDR3 sequence described in 71 differs by no more than 3 amino acid additions, deletions or substitutions of HCDR3; y) HCDR1 differs from the HCDR1 sequence described in SEQ ID NO: 50 by no more than 3 amino acid additions, deletions or substitutions. ; differs from the HCDR2 sequence described in SEQ ID NO: 61 by no more than 3 amino acid additions, deletions or deletions HCDR2; and HCDR3 which differs from the HCDR3 sequence set forth in SEQ ID NO: 72 by no more than 3 amino acid additions, deletions or substitutions; z) differs from the HCDR1 sequence described in SEQ ID NO: 148 by no more than 3 amines HCDR1 with addition, deletion or substitution of a base acid; HCDR2 differing from the HCDR2 sequence described in SEQ ID NO: 151 by no more than 3 amino acid additions, deletions or substitutions; and difference from the HCDR3 sequence described in SEQ ID NO: 154 No more than 3 amino acid additions, deletions or substitutions of HCDR3; aa) HCDR1 differing from the HCDR1 sequence described in SEQ ID NO: 149 by no more than 3 amino acid additions, deletions or substitutions; and SEQ ID NO: 152 The HCDR2 sequence differs by no more than 3 amino acid additions, deletions or substitutions of HCDR2; and HCDR3 differing from the HCDR3 sequence set forth in SEQ ID NO: 155 by no more than 3 amino acid additions, deletions or substitutions; Bb) HCDR1 differing from the HCDR1 sequence set forth in SEQ ID NO: 150 by no more than 3 amino acid additions, deletions or substitutions; no more than 3 amino acid additions to the HCDR2 sequence set forth in SEQ ID NO: 153, Deletion or substitution of HCDR2; and no more than the HCDR3 sequence described in SEQ ID NO: 156 3 amino acid added, deleted or substituted HCDR3.

Preferred ST2 antigen binding proteins of the third aspect include those comprising the light chain variable domain of a) and the heavy chain variable domain of o); they comprise the light chain variable domain of b) and a heavy chain variable domain; those comprising the light chain variable domain of c) and the heavy chain variable domain of q); they comprise the light chain variable domain of d) and r) Heavy chain variable domains; those comprising the light chain variable domain of e) and the heavy chain variable domain of s); they comprise the light chain variable domain of f) and the heavy chain of t) Variable domain; those comprising the light chain variable domain of g) and the heavy chain variable domain of u); they comprise the light chain variable domain of h) and the heavy chain of v) Domains; those comprising the light chain variable domain of i) and the heavy chain variable domain of w); they comprise the light chain variable domain of j) and the heavy chain variable domain of x) Those comprising the light chain variable domain of k) and the heavy chain variable domain of y); those comprising the light chain variable domain of l) and the heavy chain variable domain of z); Those comprising the light chain variable domain of m) and the heavy chain variable domain of aa) And their light chain variable domain comprising n), and bb) the heavy chain variable domain persons.

In a fourth aspect of the invention, the ST2 antigen-binding protein of the first, second or third aspect binds human ST2 with an affinity of less than or equal to 1 x 10 ^-10 M.

In a fifth aspect of the invention, the first, second, third or fourth aspect of the ST2 antigen binding protein inhibits human ST2 binding to human IL-33.

In a sixth aspect of the invention, the first, second, third, fourth or fifth aspect of the ST2 antigen binding protein reduces human IL-33 mediated ST2 signaling in human ST2 expressing cells.

In a seventh aspect of the invention, the sixth aspect of the ST2 antigen binding protein reduces IL-33 mediated cynomolgus ST2 signaling in cynomolgus monkey ST2 expressing cells.

In an eighth aspect of the invention, the first, second, third, fourth, fifth, sixth or seventh aspect of the ST2 antigen binding protein antibody, such as a human antibody. Preferred antibodies include those comprising a light chain having the amino acid sequence set forth in SEQ ID: 84 and having the SEQ ID NO: an antibody to the heavy chain of the amino acid sequence of 18; which comprises a light chain having the amino acid sequence set forth in SEQ ID: 85 and having the amino acid sequence set forth in SEQ ID NO: a heavy chain; those comprising a light chain having the amino acid sequence set forth in SEQ ID: 86 and a heavy chain having the amino acid sequence set forth in SEQ ID NO: 20; these include those having SEQ ID: 87 a light chain of the amino acid sequence and a heavy chain having the amino acid sequence set forth in SEQ ID NO: 21; these comprise a light chain having the amino acid sequence set forth in SEQ ID: 88 and having the SEQ ID a heavy chain of the amino acid sequence of NO: 22; those comprising a light chain having the amino acid sequence set forth in SEQ ID: 89 and a heavy chain having the amino acid sequence set forth in SEQ ID NO: Those comprising a light chain having the amino acid sequence set forth in SEQ ID: 90 and a heavy chain having the amino acid sequence set forth in SEQ ID NO: 24; these comprise having the SEQ ID: 91 a light chain of an amino acid sequence and a heavy chain having the amino acid sequence set forth in SEQ ID NO: 25; these comprise a light chain having the amino acid sequence set forth in SEQ ID: 92 and having the SEQ ID NO: the heavy chain of the amino acid sequence of 26; those comprising a light chain having the amino acid sequence set forth in SEQ ID: 93 and a heavy chain having the amino acid sequence set forth in SEQ ID NO: Those comprising a light chain having the amino acid sequence set forth in SEQ ID: 94 and a heavy chain having the amino acid sequence set forth in SEQ ID NO: 28; these include those described in SEQ ID: 160 a light chain of an amino acid sequence and a heavy chain having the amino acid sequence set forth in SEQ ID NO: 142; these comprise a light chain having the amino acid sequence set forth in SEQ ID: 161 and having SEQ ID NO: a heavy chain of the amino acid sequence of 143; and those comprising a light chain having the amino acid sequence set forth in SEQ ID: 162 and a heavy chain having the amino acid sequence set forth in SEQ ID NO: 144 .

In a ninth aspect, the invention provides an isolated nucleic acid encoding one or more polypeptide components (eg, an antibody light chain or an antibody heavy chain) of an ST2 antigen binding protein. In a preferred embodiment, the nucleic acid encodes a polypeptide comprising: a) a light chain variable domain with SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 163, SEQ ID NO: 164 or SEQ ID NO: 165 The base acid sequence has at least 95% identity; b) the heavy chain variable domain with SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 145, SEQ ID NO: 146 or SEQ The amino acid sequence set forth in ID NO: 147 has at least 95% identity; c) with SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 163, SEQ ID NO: 164 or SEQ ID NO: 165 wherein the amino acid sequence differs by no more than 5 amino acid addition, deletion or substitution of the light chain variable domain; d) and SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO :31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 The amino acid sequence described in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 145, SEQ ID NO: 146 or SEQ ID NO: 147 differs by no more than 5 amino acids Addition, deletion or substitution of a heavy chain variable domain; e) a light chain variable domain comprising: i) no more than 3 amino acid additions, deletions or differences from the LCDR1 sequence set forth in SEQ ID NO:106 Substituted LCDR1; LCDR2 which differs from the LCDR2 sequence described in SEQ ID NO: 117 by no more than 3 amino acid additions, deletions or substitutions; and no more than 3 amine groups from the LCDR3 sequence described in SEQ ID NO: 128 Acid-added, deleted or substituted LCDR3; ii) LCDR1 which differs from the LCDR1 sequence described in SEQ ID NO: 107 by no more than 3 amino acid additions, deletions or substitutions; and the LCDR2 sequence described in SEQ ID NO: 118 LCDR2 differing by no more than 3 amino acid additions, deletions or substitutions; and LCDR3 differing from the LCDR3 sequence described in SEQ ID NO: 129 by no more than 3 amino acid additions, deletions or substitutions; iii) and SEQ ID NO The LCDR1 sequence of 108 is different from the addition, deletion or substitution of 3 amino acid LCDR1; and the LCDR2 sequence described in SEQ ID NO: 119 is not more than 3 amino acid addition, deletion or substitution of LCDR2; And LCDR3 which differs from the LCDR3 sequence described in SEQ ID NO: 130 by no more than 3 amino acid additions, deletions or substitutions; iv) differs from the LCDR1 sequence described in SEQ ID NO: 109 by no more than 3 amino acid additions , missing or substituted LCDR1; LCDR2 which differs from the LCDR2 sequence described in SEQ ID NO: 120 by no more than 3 amino acid additions, deletions or substitutions; and differs from the LCDR3 sequence described in SEQ ID NO: 131 by no more than 3 LCDR3 with an amino acid added, deleted or substituted; v) LCDR1 differing from the LCDR1 sequence described in SEQ ID NO: 110 by no more than 3 amino acid additions, deletions or substitutions; and as described in SEQ ID NO: 121 LCDR2 sequence differs by no more than 3 amino acid additions, deletions or substitutions of LCDR2; and The LCDR3 sequence described in ID NO: 132 differs by no more than 3 amino acid additions, deletions or substitutions of LCDR3; vi) differs from the LCDR1 sequence described in SEQ ID NO: 111 by no more than 3 amino acid additions, deletions or Substituted LCDR1; LCDR2 which differs from the LCDR2 sequence described in SEQ ID NO: 122 by no more than 3 amino acid additions, deletions or substitutions; and no more than 3 amine groups from the LCDR3 sequence described in SEQ ID NO: 133 Acid-added, deleted or substituted LCDR3; vii) differs from the LCDR1 sequence described in SEQ ID NO: 112 by no more than 3 amino acid additions, deletions or substitutions of LCDR1; differs from the LCDR2 sequence described in SEQ ID NO: 123 LCDR2 with no more than 3 amino acid additions, deletions or substitutions; and SEQ ID The LCDR3 sequence described in NO: 134 differs by no more than 3 amino acid additions, deletions or substitutions of LCDR3; viii) differs from the LCDR1 sequence described in SEQ ID NO: 113 by no more than 3 amino acid additions, deletions or substitutions LCDR1; LCDR2 which differs from the LCDR2 sequence described in SEQ ID NO: 124 by no more than 3 amino acid additions, deletions or substitutions; and no more than 3 amino acids differs from the LCDR3 sequence described in SEQ ID NO: 135 The added, deleted or substituted LCDR3; ix) differs from the LCDR1 sequence described in SEQ ID NO: 114 by no more than 3 amino acid additions, deletions or substitutions of LCDR1; unlike the LCDR2 sequence described in SEQ ID NO: 125 LCDR2 with more than 3 amino acid additions, deletions or substitutions; and LCDR3 which differs from the LCDR3 sequence described in SEQ ID NO: 136 by no more than 3 amino acid additions, deletions or substitutions; x) and SEQ ID NO: 115 The LCDR1 sequence differs by no more than 3 amino acid additions, deletions or substitutions of LCDR1; and the LCDR2 sequence described in SEQ ID NO: 126 differs by no more than 3 amino acid additions, deletions or substitutions of LCDR2; The LCDR3 sequence described in SEQ ID NO: 137 differs by no more than 3 amino acid additions, deletions Substituted LCDR3; xi) LCDR1 differing from the LCDR1 sequence described in SEQ ID NO: 116 by no more than 3 amino acid additions, deletions or substitutions; no more than 3 amines from the LCDR2 sequence described in SEQ ID NO: 127 LCDR2 with addition, deletion or substitution of a base acid; and LCDR3 having no more than 3 amino acid additions, deletions or substitutions to the LCDR3 sequence described in SEQ ID NO: 138; xii) and LCDR1 as described in SEQ ID NO: 166 LCDR1 having a sequence difference of no more than 3 amino acid additions, deletions or substitutions; LCDR2 differing from the LCDR2 sequence described in SEQ ID NO: 169 by no more than 3 amino acid additions, deletions or substitutions; and SEQ ID NO: The LCDR3 sequence described in 172 differs by no more than 3 amino acid additions, deletions or substitutions. LCDR3; xiii) LCDR1 differing from the LCDR1 sequence described in SEQ ID NO: 167 by no more than 3 amino acid additions, deletions or substitutions; no more than 3 amine groups from the LCDR2 sequence described in SEQ ID NO: 170 Acid-added, deleted or substituted LCDR2; and LCDR3 which differs from the LCDR3 sequence described in SEQ ID NO: 173 by no more than 3 amino acid additions, deletions or substitutions; or xiv) and LCDR1 as described in SEQ ID NO: 168 LCDR1 having a sequence difference of no more than 3 amino acid additions, deletions or substitutions; LCDR2 differing from the LCDR2 sequence described in SEQ ID NO: 171 by no more than 3 amino acid additions, deletions or substitutions; and SEQ ID NO: The LCDR3 sequence described in 174 differs by no more than 3 amino acid additions, deletions or substitutions of LCDR3; or f) comprises the following heavy chain variable domains: i) differs from the HCDR1 sequence set forth in SEQ ID NO: 40 HCDR1 with more than 3 amino acid additions, deletions or substitutions; HCDR2 differing from the HCDR2 sequence described in SEQ ID NO: 51 by no more than 3 amino acid additions, deletions or substitutions; and with SEQ ID NO: 62 Said HCDR3 sequence differs by no more than 3 amino acid additions, deletions or substitutions of HCDR3; ii) and SEQ ID NO The HCDR1 sequence described in 41 differs by no more than 3 amino acid additions, deletions or substitutions of HCDR1; HCDR2 differs from the HCDR2 sequence described in SEQ ID NO: 52 by no more than 3 amino acid additions, deletions or substitutions; HCDR3 differing from the HCDR3 sequence set forth in SEQ ID NO: 63 by no more than 3 amino acid additions, deletions or substitutions; iii) differing from the HCDR1 sequence set forth in SEQ ID NO: 42 by no more than 3 amino acid additions, Deletion or substitution of HCDR1; no more than 3 amino acid additions, deletions or substitutions of HCDR2 as described in SEQ ID NO: 53; and no more than 3 HCDR3 sequences as described in SEQ ID NO: 64 Amino acid addition, deletion or substitution HCDR3; iv) HCDR1 differing from the HCDR1 sequence set forth in SEQ ID NO: 43 by no more than 3 amino acid additions, deletions or substitutions; no more than 3 amine groups differing from the HCDR2 sequence set forth in SEQ ID NO: 54 Acid-added, deleted or substituted HCDR2; and HCDR3 differing from the HCDR3 sequence set forth in SEQ ID NO: 65 by no more than 3 amino acid additions, deletions or substitutions; v) and the HCDR1 sequence set forth in SEQ ID NO: 44 HCDR1 differing by no more than 3 amino acid additions, deletions or substitutions; HCDR2 differing from the HCDR2 sequence described in SEQ ID NO: 55 by no more than 3 amino acid additions, deletions or substitutions; and SEQ ID NO: 66 The HCDR3 sequence differs by no more than 3 amino acid additions, deletions or substitutions of HCDR3; vi) HCDR1 differing from the HCDR1 sequence described in SEQ ID NO: 45 by no more than 3 amino acid additions, deletions or substitutions; HCDR2 differing from the HCDR2 sequence set forth in SEQ ID NO: 56 by no more than 3 amino acid additions, deletions or substitutions; and no more than 3 amino acid additions or deletions compared to the HCDR3 sequence set forth in SEQ ID NO:67 Or substituted HCDR3; vii) differs from the HCDR1 sequence set forth in SEQ ID NO: 46 by no more than 3 amino acid additions Addition, deletion or substitution of HCDR1; HCDR2 differing from the HCDR2 sequence set forth in SEQ ID NO: 57 by no more than 3 amino acid additions, deletions or substitutions; and no more than the HCDR3 sequence described in SEQ ID NO: 68 3 amino acid added, deleted or substituted HCDR3; viii) HCDR1 differing from the HCDR1 sequence described in SEQ ID NO: 47 by no more than 3 amino acid additions, deletions or substitutions; and SEQ ID NO: 58 Said HCDR2 sequence differs by no more than 3 amino acid additions, deletions or substitutions of HCDR2; and HCDR3 which differs from the HCDR3 sequence set forth in SEQ ID NO: 69 by no more than 3 amino acid additions, deletions or substitutions; Ix) HCDR1 differing from the HCDR1 sequence set forth in SEQ ID NO: 48 by no more than 3 amino acid additions, deletions or substitutions; no more than 3 amino acid additions to the HCDR2 sequence set forth in SEQ ID NO: 59, a deleted or substituted HCDR2; and an HCDR3 which differs from the HCDR3 sequence set forth in SEQ ID NO: 70 by no more than 3 amino acid additions, deletions or substitutions; x) differs from the HCDR1 sequence described in SEQ ID NO: 49 by no more than HCDR1 added, deleted or substituted with 3 amino acids; HCDR2 differing from the HCDR2 sequence described in SEQ ID NO: 60 by no more than 3 amino acid additions, deletions or substitutions; and as described in SEQ ID NO: 71 HCDR3 sequences differ by no more than 3 amino acid additions, deletions or substitutions of HCDR3; xi) HCDR1 differing from the HCDR1 sequence set forth in SEQ ID NO: 50 by no more than 3 amino acid additions, deletions or substitutions; The HCDR2 sequence described in NO: 61 differs by no more than 3 amino acid additions, deletions or substitutions of HCDR2; and the HCDR3 sequence described in SEQ ID NO: 72 differs by no more than 3 amino acid additions, deletions or substitutions. HCDR3;xii) differs from the HCDR1 sequence described in SEQ ID NO: 148 by no more than 3 amino acid additions, deficiency Or substituted HCDR1; HCDR2 differing from the HCDR2 sequence set forth in SEQ ID NO: 151 by no more than 3 amino acid additions, deletions or substitutions; and no more than 3 amines from the HCDR3 sequence set forth in SEQ ID NO: 154 HCDR3 added, deleted or substituted by a base acid; xiii) HCDR1 differing from the HCDR1 sequence set forth in SEQ ID NO: 149 by no more than 3 amino acid additions, deletions or substitutions; and HCDR2 sequences as described in SEQ ID NO: 152 HCDR2 differing by no more than 3 amino acid additions, deletions or substitutions; and HCDR3 differing from the HCDR3 sequence described in SEQ ID NO: 155 by no more than 3 amino acid additions, deletions or substitutions; or xiv) and SEQ ID The HCDR1 sequence described in NO: 150 differs by no more than 3 amine groups. Acid-added, deleted or substituted HCDR1; HCDR2 differing from the HCDR2 sequence described in SEQ ID NO: 153 by no more than 3 amino acid additions, deletions or substitutions; and the HCDR3 sequence described in SEQ ID NO: 156 HCDR3 with more than 3 amino acids added, deleted or substituted.

In certain embodiments of the ninth aspect, the polypeptide encodes an antibody light chain and is SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 157, SEQ ID NO: 158 or SEQ ID NO The nucleotide sequence of: 159 is at least 80%, at least 90%, at least 95% or 100% identical. In other embodiments of the ninth aspect, the polypeptide encodes an antibody heavy chain and is SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 139, SEQ ID NO: 140 or SEQ ID NO: The nucleotide sequence set forth in 141 is at least 80%, at least 90%, at least 95% or 100% identical.

In a tenth aspect, the invention provides a performance vector comprising one or more isolated nucleic acids of the eighth aspect. In certain embodiments, the expression vector encodes an antibody light chain, an antibody heavy chain, or both an antibody light chain and a heavy chain.

In an eleventh aspect, the invention provides a recombinant host cell comprising one or more isolated nucleic acids operably linked to a promoter, comprising one or more of the tenth aspect of the invention A recombinant host cell that expresses a vector. In a preferred embodiment, the recombinant host cell secretes an antibody that binds to ST2. Preferred host cell lines are mammalian host cells, including CHO cell lines.

In a twelfth aspect, the invention provides a method of treating an autoimmune or inflammatory condition, the method comprising administering to a patient in need thereof a therapeutically effective amount of the first, second, third, fourth, fifth ST2 antigen-binding egg of any of the sixth, seventh or eighth aspects White matter. In a preferred embodiment, the ST2 antigen binding protein line comprises a light chain variable domain amino acid sequence as set forth in SEQ ID NO: 95 and a heavy chain variable domain amino group as set forth in SEQ ID NO: An antibody to an acid sequence (eg, Ab1) comprising a light chain variable domain amino acid sequence as set forth in SEQ ID NO: 96 and a heavy chain variable domain amino acid sequence as set forth in SEQ ID NO: Antibody (eg, Ab2), an antibody comprising a light chain variable domain amino acid sequence as set forth in SEQ ID NO: 97 and a heavy chain variable domain amino acid sequence as set forth in SEQ ID NO: 31 (eg, Ab3), an antibody comprising a light chain variable domain amino acid sequence as set forth in SEQ ID NO: 98 and a heavy chain variable domain amino acid sequence as set forth in SEQ ID NO: 32 (eg, , Ab4), an antibody comprising a light chain variable domain amino acid sequence as set forth in SEQ ID NO: 99 and a heavy chain variable domain amino acid sequence as set forth in SEQ ID NO: 33 (eg, Ab5) An antibody comprising a light chain variable domain amino acid sequence as set forth in SEQ ID NO: 100 and a heavy chain variable domain amino acid sequence as set forth in SEQ ID NO: 34 (eg, A B6) an antibody comprising a light chain variable domain amino acid sequence as set forth in SEQ ID NO: 101 and a heavy chain variable domain amino acid sequence as set forth in SEQ ID NO: 35 (eg, Ab7) An antibody (eg, Ab8) comprising a light chain variable domain amino acid sequence as set forth in SEQ ID NO: 102 and a heavy chain variable domain amino acid sequence as set forth in SEQ ID NO: 36, comprising An antibody (eg, Ab9), such as SEQ as described in SEQ ID NO: 103, of the light chain variable domain amino acid sequence and the heavy chain variable domain amino acid sequence set forth in SEQ ID NO: 37 The light chain variable domain amino acid sequence of ID NO: 104 and the antibody (eg, Ab10) of the heavy chain variable domain amino acid sequence set forth in SEQ ID NO: 38, comprising SEQ ID NO The light chain variable domain amino acid sequence of 105 and the antibody (eg, Ab11) of the heavy chain variable domain amino acid sequence set forth in SEQ ID NO: 39; comprising SEQ ID NO: 163 The light chain variable domain amino acid sequence and the antibody (eg, Ab30) of the heavy chain variable domain amino acid sequence set forth in SEQ ID NO: 45; comprising as set forth in SEQ ID NO: 164 A light chain variable domain and the amino acid sequence as SEQ ID NO: the amino acid sequence of the variable domain of the heavy chain 146 antibodies (e.g., Ab32); or an antibody comprising a light chain variable domain amino acid sequence as set forth in SEQ ID NO: 165 and a heavy chain variable domain amino acid sequence as set forth in SEQ ID NO: 147 (eg, Ab33) ). In a preferred embodiment, the ST2 antigen binding protein inhibits IL-33 binding to ST2. In a particularly preferred embodiment, the autoimmune or inflammatory condition is asthma, inflammatory bowel disease, rheumatoid arthritis, psoriasis, atopic dermatitis, fibrosis, chronic obstructive pulmonary disease, systemic lupus erythematosus, sclerosis, Wagner's granuloma, Beth's disease, sinusitis, nasal polyps, eosinophilic bronchitis and cardiovascular disease.

In a thirteenth aspect, the present invention provides the preparation of the first, second, third, fourth, fifth, by culturing the recombinant host cell of the eleventh aspect and isolating the ST2 antigen binding protein from the culture. A method of binding a protein to an ST2 antigen of any of the sixth, seventh or eighth aspects.

In a fourteenth aspect, the invention provides any one of the first, second, third, fourth, fifth, sixth, seventh or eighth aspect of the antibody cross-linked to an antibody selected from the group consisting of Competing ST2 antigen binding protein: a) an antibody comprising: a light chain comprising the amino acid sequence set forth in SEQ ID: 84 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 18; b) comprising An antibody comprising: a light chain comprising the amino acid sequence set forth in SEQ ID: 85 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19; c) an antibody comprising: SEQ ID: 86 a light chain of the amino acid sequence and a heavy chain comprising the amino acid sequence of SEQ ID NO: 20; d) comprising an antibody comprising: a light chain comprising the amino acid sequence of SEQ ID: 87 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 21; e) an antibody comprising: a light chain comprising the amino acid sequence set forth in SEQ ID: 88 and comprising the amine of SEQ ID NO: a heavy chain of a base acid sequence; f) an antibody comprising: a light chain comprising the amino acid sequence set forth in SEQ ID: 89 and comprising the amino acid of SEQ ID NO: Column of the heavy chain; g) an antibody comprising: a light chain comprising the amino acid sequence set forth in SEQ ID: 90 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 24; h) an antibody comprising: SEQ ID a light chain of the amino acid sequence of 91 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 25; i) an antibody comprising: the amino acid sequence of SEQ ID: 92 a light chain and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 26; j) an antibody comprising: a light chain comprising the amino acid sequence set forth in SEQ ID: 93 and comprising SEQ ID NO: The heavy chain of the amino acid sequence; k) comprises an antibody comprising: a light chain comprising the amino acid sequence set forth in SEQ ID: 94 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 28; l) an antibody comprising: a light chain comprising the amino acid sequence set forth in SEQ ID: 160 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 142; m) an antibody comprising: SEQ ID a light chain of the amino acid sequence of 161 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 143; n) comprising an antibody comprising: SEQ ID: 162 And the light chain comprises the amino acid sequence of SEQ ID NO: 144 in the heavy chain amino acid sequences.

In a fifteenth aspect, the invention provides an isolated ST2 antigen binding protein, preferably an antibody or antigen-binding fragment thereof, comprising a polypeptide comprising human ST2 domain 1 and domain 2 (SEQ ID NO: 175), wherein When a single mutation is introduced into the human ST2 domain 1 or domain 2 of the polypeptide, the binding is significantly inhibited, wherein the single mutation is selected from the group consisting of L14R, I15R, S33R, E43R, V47R, A62R, G65R, T79R, D92R, D97R, V104R, G138R, N152R and V176R. "Significantly inhibited" means that the measured binding difference is statistically significant. In a preferred embodiment, binding is significantly inhibited for two or more members of the population, including all members of the population. In certain embodiments of the fifteenth aspect, when a single mutation is introduced into human ST2 domain 1 or domain 2 of the polypeptide, binding is also significantly activated, wherein the single The mutation is selected from the group consisting of L53R, R72A and S73R. "Significantly activated" means that the measured binding difference is statistically significant. In a preferred embodiment, the binding is significantly activated for all members of the population. In certain embodiments of the fifteenth aspect, the ST2 binding protein is associated with a heavy chain comprising a light chain comprising the amino acid sequence set forth in SEQ ID: 85 and comprising the amino acid sequence set forth in SEQ ID NO: The antibody cross-competes in combination with human ST2. In a particularly preferred embodiment, the antigen binding protein is an antigen binding protein of the first, second, third, fourth, fifth, sixth, seventh or eighth aspect.

In a sixteenth aspect, the present invention provides an antigen-binding protein of the first, second, third, fourth, fifth, sixth, seventh, eighth, fourteenth or fifteenth aspect, wherein The ST2 binding protein, preferably the antibody or antigen binding fragment thereof, binds to a portion of ST2 comprising amino acids 33-44 and/or 88-94 of SEQ ID NO: 1, as determined by hydrogen/deuterium exchange analysis.

In a seventeenth aspect, the present invention provides an antigen of the first, second, third, fourth, fifth, sixth, seventh, eighth, fourteenth, fifteenth or sixteenth aspect A binding protein, preferably an antibody or antigen-binding fragment thereof, which binds to ST2 to create an interface, wherein the interface resulting from the binding comprises an ST2 residue selected from the group consisting of K1, F2, P19, R20, Q21, G22 , K23, Y26, I70, V71, R72, S73, P74, T75, F76, N77, R78, T79 and Y81. In a preferred embodiment of the seventeenth aspect, the interface resulting from the binding comprises ST2 residues P19, R20, Q21, G22, K23 and/or Y26; ST2 residues I70, V71, R72, S73, P74, T75, F76, N77, R78, T79 and/or Y81; or ST2 residues P19, R20, Q21, G22, K23, Y26, I70, V71, R72, S73, P74, T75, F76, N77, R78, T79 and Y81. The interface can be determined by combining the difference in solvent exposure between ST2 and unbound ST2 and the interfacial residues are defined as those amino acids with a difference greater than 10% and those that form a water-mediated hydrogen bond with the antibody It is determined by the amino acid having at least one atom within 5 Å of the antibody.

In an eighteenth aspect, the invention provides an isolated ST2 antigen binding protein, preferably an antibody or antigen binding fragment thereof comprising: a) at least 90% of the amino acid sequence described in SEQ ID NO: 96 or a light chain variable domain of at least 95% identity; b) a heavy chain variable domain having at least 90% or at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 30; c) a) a light chain variable domain and a heavy chain variable domain of b); d) differs from the amino acid sequence set forth in SEQ ID NO: 96 by no more than 10 or no more than 5 amino acid additions, deletions or a substituted light chain variable domain; e) a heavy chain variable domain that differs from the amino acid sequence set forth in SEQ ID NO: 30 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions; f) the heavy chain variable domain of d) the light chain variable domain e); g) the light chain variable domain comprising: no more than 3 amines from the LCDR1 sequence described in SEQ ID NO: 107 LCDR1 added, deleted or substituted with a base acid, LCDR2, which differs from the LCDR2 sequence described in SEQ ID NO: 118 by no more than 3 amino acid additions, deletions or substitutions, and SEQ ID NO: 129 The LCDR3 sequence differs by no more than 3 amino acid additions, deletions or substitutions of LCDR3; h) comprises a heavy chain variable domain that differs from the HCDR1 sequence described in SEQ ID NO: 41 by no more than 3 amines HCDR1 added, deleted or substituted by a base acid, HCDR2 which differs from the HCDR2 sequence described in SEQ ID NO: 52 by no more than 3 amino acid additions, deletions or substitutions, and which differs from the HCDR3 sequence described in SEQ ID NO: 63 No more than 3 amino acid added, deleted or substituted HCDR3; or i) g) light chain variable domain and h) heavy chain variable domain.

In a preferred embodiment of the eighteenth aspect, the light chain variable region comprises D28 or a conservative substitution thereof, I29 or a conservative substitution thereof, S30 or a conservative substitution thereof, N31 or a conservative substitution thereof, Y32 or a conservative substitution thereof, Y49 Or a conservative substitution thereof, D50 or a conservative substitution thereof, N53 or a conservative substitution thereof, E55 or a conservative substitution thereof, T56 or a conservative substitution thereof, D91 or a conservative substitution thereof, D92 or a conservative substitution thereof, N93 or a conservative substitution thereof, F94 or Conservative substitution or L96 or its conservative substitution. In other preferred embodiments, the light chain variable region comprises D28 or a conservative substitution thereof, N31 or a conservative substitution thereof, D50 or a conservative substitution thereof, N53 or a conservative substitution thereof, E55 Or its conservative substitution, D91 or its conservative substitution and D92 or its conservative substitution. In other preferred embodiments, the light chain variable region comprises D28, N31, D50, N53, E55, D91, and D92.

The eighteenth aspect also includes an ST2 binding protein, preferably an antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises W33 or a conservative substitution thereof, I50 or a conservative substitution thereof, D57 or a conservative substitution thereof, R59 or a conservative thereof a substitution, H99 or a conservative substitution thereof, G100 or a conservative substitution thereof, T101 or a conservative substitution thereof, S102 or a conservative substitution thereof, S103 or a conservative substitution thereof, D104 or a conservative substitution thereof, Y105 or a conservative substitution thereof, or Y106 or a conservative substitution thereof Wherein the heavy chain variable region comprises S102 or a conservative substitution thereof, S103 or a conservative substitution thereof, D104 or a conservative substitution thereof, and Y105 or a conservative substitution thereof; and wherein the heavy chain variable region comprises S102, S103, D104 and Y105.

In certain embodiments of the eighteenth aspect, the ST2 antigen binding protein specifically binds to human ST2 with an affinity of less than or equal to 1 x 10 ^-10 M, inhibits human ST2 binding to human IL-33, and reduces human ST2 expression in cells Human IL-33-mediated ST2 signaling, inhibition of cynomolgus monkey ST2 binding to cynomolgus IL-33, reducing IL-33-mediated cynomolgus ST2 signaling in cynomolgus ST2 expressing cells, and/or An antibody, such as a human antibody.

Figure 1 ST2 mAb treatment significantly inhibited IL-33-induced IL-5 in the branched tracheal alveolar lavage fluid (BALF) Balb/c and C57B1/6 mice.

Figure 2 ST2 mAb treatment is effective in a sputum allergen (CRA)-induced asthma model. ST2 antibody treated mice had significantly fewer BALF eosinophilic leukocytes than isotype control Ig treated mice.

Figure 3. Inhibition of human IL-33-induced IL-5 production by ST2 mAbs from various donor CD4+ T cells. The (-) line depicts positive control values for the combination of human IL-33 and human IL-2 without inhibition. (....) shows the positive control value of human IL-2. The (--) line shows the medium control value.

Figure 4 Dose response of human IL-33 in human NK cell analysis.

Figure 5. Reduction of IL-33 activity by Ab2 against commercially available ST2 antibodies in human NK cell assays. Pure lines HB12, FB9 and 2A5 are available from MBL International. Pure B4E6 lines were obtained from MD Biosciences. Pure 97203 is available from R&D Systems.

Figure 6 Positioning of the region of Ab2 binding in ST2 as determined by HDX (see Example 12). The region of the amino acid 15-26 corresponding to the extracellular domain of ST2 is highlighted in red and the region of the amino acid 70-76 corresponding to the extracellular domain of ST2 is highlighted as magenta.

Figure 7. Overall structure of the ST2/Ab2 sc-dsFv complex. The two Ab2 sc-dsFv molecules are shown as a cartoon representation and the light chain (LC)/heavy chain (HC) pairs are cyan/blue or light yellow/gold, respectively. The two ST2 molecules are shown as magenta and green cartoons.

Figure 8 combines the interface. ST2 is shown as a yellow cartoon. The heavy and light chains of Ab2 are shown in gray and wheat cartoons. The CDR loops of the heavy and light chains are colored in the following order: CDR1: red (HC) or light red (LC); CDR2: green (HC) or light green (LC); and CDR3: blue (HC) or light blue Color (LC).

Figure 9. Electrostatic surface potential plot of ST2 and Ab2 sc-dsFv. Figure 9A). Charge and surface complementarity of ST2 and Ab2 sc-dsFv. The bonding interface is highlighted in the ring. Figure 9B) Left: Ab2 (grey/wheat cartoon) combined with a positively charged patch on ST2 (surface); right: ST2 (yellow cartoon) combined with an acidic patch of Ab2 sc-dsFv (surface). For an electrostatic potential map, the red surface represents a negative charge and the blue surface represents a positive charge.

Figure 10 Residues that form an interface with the antigen when bound to ST2 in the Ab2 variable domain. The CDR area is framed. Residues within the interface are shown in bold. The residue which forms a hydrogen bond or a salt bridge with the amino acid in ST2 is italic.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter. All references cited in the text of this specification are hereby expressly incorporated by reference.

Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, tissue culture and transformation, protein purification, and the like. The enzymatic reaction and purification techniques can be carried out according to the manufacturer's instructions, or as practiced in the art, or as described herein. The following procedures and techniques are generally carried out in accordance with the conventional methods well known in the art and as described in the various general and more specific references cited and discussed in the specification. See, for example, Sambrook et al, 2001, Molecular Cloning: A Laboratory Manuel , 3rd edition, Cold Spring Harbor Laboratory Press, cold Spring Harbor, NY, which is incorporated herein by reference for all purposes. Unless specific definitions are provided, the nomenclature used in analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry as described herein, as well as their laboratory procedures and techniques, are well known and employed in the art. Standard techniques are available for chemical synthesis; chemical analysis; pharmaceutical preparation, formulation and delivery; and patient treatment.

ST2

The antigen binding proteins described herein bind to ST2. ST2 behaves as both a soluble non-signaling variant (soluble ST2 or sST2) and a full-length transmembrane form (FL ST2, ST2 or ST2L). Exemplary human ST2L amino acid sequences are provided herein in Table 1. The protein consists of several domains: amino acids 1-18 correspond to cleavable pre-sequences during protein processing in mammalian cells; amino acids 19-331 correspond to extracellular domains; amino acids 332- 350 corresponds to a transmembrane domain; and amino acids 351-556 correspond to intracellular domains. In a preferred embodiment, the antigen binding protein binds to the extracellular domain of ST2L and prevents the interaction of ST2 with IL-33. Exemplary human IL-33 amino acid sequences are provided in Table 1.

IL-33 signals via a heterodimeric receptor comprising ST2L and AcP. Exemplary human AcP amino acid sequences are provided in Table 1. This protein also consists of several domains: amino acids 1-20 correspond to cleavable pre-sequences during protein processing in mammalian cells; amino acids 21-367 correspond to extracellular domains; amino acids 368 -388 corresponds to the transmembrane domain; and the amino acids 389-570 correspond to the intracellular domain. In an example embodiment In this, the ST2 antigen binding protein binds to ST2L and blocks IL-33 mediated signaling in cells expressing ST2L and AcP.

An exemplary embodiment of the invention binds both human ST2 and cynomolgus ST2 with high affinity, including those that bind cynomolgus IL-33 and cynomolgus ST2 with high affinity and block their interaction. These properties allow for information toxicology studies in non-human primates.

Exemplary amino acid sequences of cynomolgus monkey ST2L are provided in Table 2. The protein consists of several domains: amino acids 1-18 correspond to cleavable pre-sequences during protein processing in mammalian cells; amino acids 19-331 correspond to extracellular domains; amino acids 332- 350 corresponds to a transmembrane domain; and amino acids 351-556 correspond to intracellular domains.

Exemplary amino acid sequences of cynomolgus monkey AcP are provided in Table 2. The protein consists of several domains: amino acids 1-20 correspond to cleavable pre-sequences during protein processing in mammalian cells; amino acids 21-367 correspond to extracellular domains; amino acids 368- 388 corresponds to the transmembrane domain; and the amino acid 389-570 corresponds to the intracellular domain.

Exemplary amino acid sequences of cynomolgus IL-33 are provided in Table 2.

ST2 antigen binding protein

The present invention provides an antigen binding protein that specifically binds to ST2. Examples of antigen binding proteins comprise peptides and/or polypeptides that specifically bind to ST2. Such peptides or polypeptides may optionally include one or more post-translational modifications. Examples of antigen binding proteins include antibodies that specifically bind to ST2 and fragments thereof, as defined herein in different ways. These include antibodies that specifically bind to human ST2, including those that inhibit IL-33 binding and/or activate ST2.

The antigen-binding protein of the present invention specifically binds to ST2. As used herein, "specifically binds" means that the antigen-binding protein preferentially binds to ST2 relative to other proteins. In some embodiments, "specifically binds" means that the ST2 antigen binding protein has a higher affinity for ST2 than for other proteins. The binding affinity of the ST2 antigen binding protein that specifically binds to ST2 to human ST2 may be less than or equal to 1 x 10 ^-7 M, less than or equal to 2 x 10 ^-7 M, less than or equal to 3 x 10 ^-7 M, less than or equal to 4 ×10 ^-7 M, less than or equal to 5 × 10 ^-7 M, less than or equal to 6 × 10 ^-7 M, less than or equal to 7 × 10 ^-7 M, less than or equal to 8 × 10 ^-7 M, less than or equal to 9 ×10 ^-7 M, less than or equal to 1×10 ^-8 M, less than or equal to 2×10 ^-8 M, less than or equal to 3×10 ^-8 M, less than or equal to 4×10 ^-8 M, less than or equal to 5 ×10 ^-8 M, less than or equal to 6×10 ^-8 M, less than or equal to 7×10 ^-8 M, less than or equal to 8×10 ^-8 M, less than or equal to 9×10 ^-8 M, less than or equal to 1 ×10 ^-9 M, less than or equal to 2×10 ^-9 M, less than or equal to 3×10 ^-9 M, less than or equal to 4×10 ^-9 M, less than or equal to 5×10 ^-9 M, less than or equal to 6 ×10 ^-9 M, less than or equal to 7 × 10 ^-9 M, less than or equal to 8 × 10 ^-9 M, less than or equal to 9 × 10 ^-9 M, less than or equal to 1 × 10 ^-10 M, less than or equal to 2 ×10 ^-10 M, less than or equal to 3 × 10 ^-10 M, less than or equal to 4 × 10 ^-10 M, less than or equal to 5 × 10 ^-10 M, less than or equal to 6 × 10 ^-10 M, less than or equal to 7 × 10 ^-10 M, less than or equal to 8 × 10 ^-10 M, less than or equal to 9 × 10 ^-10 M, less than or equal to 1 × 10 ^-11 M, less than or equal to 2 × 10 ^-11 M, less than or equal to 3×10 ^-11 M, less than or equal to 4×10 ^-11 M, less than or equal to 5×10 ^-11 M, less than or equal to 6×10 ^-11 M, less than or equal to 7×10 ^-11 M, less than or equal to 8×10 ^-11 M, less than or equal to 9×10 ^-11 M, less than or equal to 1×10 ^-12 M, less than or equal to 2×10 ^-12 M, less than or equal to 3×10 ^-12 M, less than or equal to 4×10 ^-12 M, less than or equal to 5×10 ^-12 M, less than or equal to 6×10 ^-12 M, less than or equal to 7×10 ^-12 M, less than or equal to 8×10 ^-12 M or less than or equal to 9 x 10 ^-12 M.

Methods for measuring the binding affinity of antigen binding proteins are well known in the art. Methods for affinity determination include surface plasma resonance (SPR) (Morton and Myszka "Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors" Methods in Enzymology (1998) 295, 268-294), Biolayer Interferometry (Abdiche et al. Determining Kinetics and Affinities of Protein Interactions using a Parallel Real-time Label-free Biosensor, the Octet" Analytical Biochemistry (2008) 377, 209-217), KinexA Analysis (KinExA) (Darling and Braction "Kinetic exclusion assay technology: characterization of Molecular interactions" Assay and Drug Dev Tech (2004) 2, 647-657), isothermal calorimetry (Pierce et al. "Isothermal Titration Calorimetry of Protein-Protein Interactions" Methods ( 1999) 19, 213-221) and analytical ultracentrifugation (Lebowitz et al. "Modern analytical ultracentrifugation in protein science: A tutorial review" Protein Science (2002), 11: 2067-2079). Example 3 provides an example method.

It will be understood that when reference is made herein to various embodiments of ST2 binding antibodies, it also encompasses ST2 binding fragments of such antibodies. The ST2 binding fragment comprises any of the antibody fragments or domains described herein that retain the ability to specifically bind to ST2. The ST2 binding fragment can be in any of the scaffolds described herein.

In certain therapeutic embodiments, the ST2 antigen binding protein inhibits ST2 binding to IL-33 and/or inhibits one or more of the biological activities associated with ST2 binding to IL-33, for example, IL-33 mediated signaling. These antigen-binding proteins are considered to be "neutralizing". In certain embodiments, the neutralizing ST2 antigen binding protein specifically binds to ST2 and inhibits binding of ST2 to IL-33 by any value between 10% and 100%, such as at least about 20%, 21%, 22 %, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55% 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72 %, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or more. For example, ST2 antigen binding protein neutralization ability can be tested by measuring the ability of an antigen binding protein to block IL-33 binding to ST2 or IL-33 binding to co-receptors ST2 and AcP, for example, see Example 6 for IL-33 resistance. Break analysis. Alternatively, the ST2 antigen binding protein neutralization capacity can be tested in an assay that measures IL-33 mediated biological function in an assay that measures the effect of the presence of the ST2 antigen binding protein. For example, IL-33 induces the ability of a biological response (eg, intracellular signaling of a medium or increased expression of mRNA or secretion of a medium), such as from eosinophils, basophils, T Interleukins and chemokines in cells such as cells, mast cells, NK cells, NKT cells, neutrophils or innate helper cells. Alternatively, IL-33 promotes differentiation, proliferation, and survival of cells such as eosinophils, basophils, T cells, mast cells, NK cells, NKT cells, neutrophils, or innate helper cells. The ability to chemotaxis, shape change or adhesion properties. Alternatively, IL-33 induces certain cell activation markers such as CD11b in eosinophils, basophils, T cells, mast cells, NK cells, NKT cells, neutrophils or innate aids. The ability of cells to express on the cell, such as cells. Exemplary methods are provided in Examples 7-10.

Examples of antigen binding proteins comprise a scaffold structure and one or more complementarity determining regions (CDRs) as defined herein in a different manner. Embodiments further include an antigen binding protein comprising a scaffolding structure and one or more antibody heavy or light chain variable domains. Examples include antibodies comprising a light chain variable domain selected from the group consisting of: Ab1 light chain variable domain (LCv), Ab2 LCv, Ab3 LCv, Ab4 LCv, Ab5 LCv, Ab6 LCv, Ab7 LCv , Ab8 LCv, Ab9 LCv, Ab10LCv, Ab11 LCv, Ab30 LCv, Ab32 LCv and Ab33 LCv (SEQ ID NOS: 95-105, 163-165, respectively) and/or heavy chain variable structure selected from the group consisting of Domain: Ab1 heavy chain variable domain (HCv), Ab2 HCv, Ab3 HCv, Ab4 HCv, Ab5 HCv, Ab6 HCv, Ab7 HCv, Ab8 HCv, Ab9 HCv, Ab10 HCv, Ab11HCv, Ab30HCv, Ab32HCv and Ab33HCv (SEQ ID NOS: 29-39, 145-147, respectively) as well as fragments, derivatives, mutant proteins and variants thereof.

An exemplary light chain comprising Ab1 LCv comprises the light chain of the amino acid sequence set forth in SEQ ID NO:84.

An exemplary light chain comprising Ab2 LCv comprises the light chain of the amino acid sequence set forth in SEQ ID NO:85.

An exemplary light chain comprising Ab3 LCv comprises the light chain of the amino acid sequence set forth in SEQ ID NO:86.

An exemplary light chain comprising Ab4 LCv comprises the light chain of the amino acid sequence set forth in SEQ ID NO:87.

An exemplary light chain comprising Ab5 LCv comprises the light chain of the amino acid sequence set forth in SEQ ID NO:88.

An exemplary light chain comprising Ab6 LCv comprises the light chain of the amino acid sequence set forth in SEQ ID NO:89.

An exemplary light chain comprising Ab7 LCv comprises the light chain of the amino acid sequence set forth in SEQ ID NO:9O.

An exemplary light chain comprising Ab8 LCv comprises the light chain of the amino acid sequence set forth in SEQ ID NO:91.

An exemplary light chain comprising Ab9 LCv comprises the light chain of the amino acid sequence set forth in SEQ ID NO:92.

An exemplary light chain comprising Ab10 LCv comprises the light chain of the amino acid sequence set forth in SEQ ID NO:93.

An exemplary light chain comprising Ab11 LCv comprises the light chain of the amino acid sequence set forth in SEQ ID NO:94.

An exemplary light chain comprising Ab30 LCv comprises the amino acid described in SEQ ID NO: 160 The light chain of the sequence.

An exemplary light chain comprising Ab32 LCv comprises the light chain of the amino acid sequence set forth in SEQ ID NO:161.

An exemplary light chain comprising Ab33 LCv comprises the light chain of the amino acid sequence set forth in SEQ ID NO:162.

An exemplary heavy chain comprising Ab1 HCv comprises the heavy chain of the amino acid sequence set forth in SEQ ID NO: 18.

An exemplary heavy chain comprising Ab2 HCv comprises the heavy chain of the amino acid sequence set forth in SEQ ID NO: 19.

An exemplary heavy chain comprising Ab3 HCv comprises the heavy chain of the amino acid sequence set forth in SEQ ID NO:20.

An exemplary heavy chain comprising Ab4 HCv comprises the heavy chain of the amino acid sequence set forth in SEQ ID NO:21.

An exemplary heavy chain comprising Ab5 HCv comprises the heavy chain of the amino acid sequence set forth in SEQ ID NO:22.

An exemplary heavy chain comprising Ab6 HCv comprises the heavy chain of the amino acid sequence set forth in SEQ ID NO:23.

An exemplary heavy chain comprising Ab7 HCv comprises the heavy chain of the amino acid sequence set forth in SEQ ID NO: 24.

An exemplary heavy chain comprising Ab8 HCv comprises the heavy chain of the amino acid sequence set forth in SEQ ID NO:25.

An exemplary heavy chain comprising Ab9 HCv comprises the heavy chain of the amino acid sequence set forth in SEQ ID NO:26.

An exemplary heavy chain comprising Ab10 HCv comprises the heavy chain of the amino acid sequence set forth in SEQ ID NO:27.

An exemplary heavy chain comprising Ab11 HCv comprises the amino acid described in SEQ ID NO:28 The heavy chain of the sequence.

An exemplary heavy chain comprising Ab30 HCv comprises the heavy chain of the amino acid sequence set forth in SEQ ID NO:142.

An exemplary heavy chain comprising Ab32 HCv comprises the heavy chain of the amino acid sequence set forth in SEQ ID NO:143.

An exemplary heavy chain comprising Ab33 HCv comprises the heavy chain of the amino acid sequence set forth in SEQ ID NO:144.

Other examples of contemplated scaffolds include: fibronectin, neopotassium CBM4-2, apolipoprotein, T-cell receptor, protein-A domain (protein Z), Im9, TPR protein, zinc finger domain, pVIII, avian pancreatic polypeptide, GCN4, WW domain Src homology domain 3, PDZ domain, TEM-1 β-prolinease, thioredoxin, staphylococcal ribozyme, PHD-finger domain, CL- 2. BPTI, APPI, HPSTI, ecotin, LACI-D1, LDTI, MTI-II, scorpion toxin, insect defensin-A peptide, EETI-II, Min-23, CBD, PBP, cytochrome b -562, Ldl receptor domain, gamma crystal protein, ubiquitin, transferrin and or C-type lectin-like domain. Non-antibody scaffolds and their use as therapeutic agents are reviewed in Gebauer and Skerra, Curr . Opin . Chem . Biol ., 13: 245-255 (2009) and Binz et al, Nat. Biotech., 23(10): 1257- In 1268 (2005), the entire contents of each of these references are hereby incorporated by reference.

The present invention includes antibodies comprising the following variable domains: Ab1 LCv/Ab1 HCv (SEQ ID NO: 95/SEQ ID NO: 29), Ab2 LCv/Ab2 HCv (SEQ ID NO: 96/SEQ ID NO: 30) ), Ab3 LCv/Ab3 HCv (SEQ ID NO: 97/SEQ ID NO: 31), Ab4 LCv/Ab4 HCv (SEQ ID NO: 98/SEQ ID NO: 32), Ab5 LCv/Ab5 HCv (SEQ ID NO: 99/SEQ ID NO: 33), Ab6 LCv/Ab6 HCv (SEQ ID NO: 100/SEQ ID NO: 34), Ab7 LCv/Ab7 HCv (SEQ ID NO: 101/SEQ ID NO: 35), Ab8 LCv/ Ab8 HCv (SEQ ID NO: 102 / SEQ ID NO: 36), Ab9 LCv/Ab9 HCv (SEQ ID NO: 103 / SEQ ID NO: 37), Ab10 LCv/Ab10 HCv (SEQ ID NO: 104/SEQ ID NO: 38), Ab11 LCv/Ab11 HCv (SEQ ID NO: 105/SEQ ID NO: 39), Ab30 LCv/Ab30 HCv (SEQ ID NO: 163/SEQ ID NO: 145), Ab32 LCv/Ab32 HCv (SEQ ID NO: 164/SEQ ID NO: 146), Ab33 LCv/Ab33 HCv (SEQ ID NO: 165/SEQ ID NO: 147), and combinations thereof, and fragments thereof, Derivatives, mutant proteins and variants.

Exemplary antibodies of the invention include Ab1 (SEQ ID NO: 84/SEQ ID NO: 18), Ab2 (SEQ ID NO: 85/SEQ ID NO: 19), Ab3 (SEQ ID NO: 86/SEQ ID NO: 20) ), Ab4 (SEQ ID NO: 87/SEQ ID NO: 21), Ab5 (SEQ ID NO: 88/SEQ ID NO: 22), Ab6 (SEQ ID NO: 89/SEQ ID NO: 23), Ab7 (SEQ) ID NO: 90 / SEQ ID NO: 24), Ab8 (SEQ ID NO: 91 / SEQ ID NO: 25), Ab9 (SEQ ID NO: 92 / SEQ ID NO: 26), Ab10 (SEQ ID NO: 93/ SEQ ID NO: 27), Ab11 (SEQ ID NO: 94 / SEQ ID NO: 28), Ab30 (SEQ ID NO: 160 / SEQ ID NO: 142), Ab32 (SEQ ID NO: 161 / SEQ ID NO: 143 And Ab33 (SEQ ID NO: 162 / SEQ ID NO: 144).

Typically, each variable domain of an antibody light or heavy chain comprises three CDRs. The heavy chain variable domain comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3). The light chain variable domain comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3). In certain embodiments, an antigen binding protein comprises one or more CDRs contained within a preferred variable domain described herein.

Examples of such CDRs include, but are not limited to, CDRs of Ab1 LCv: LCDR1 (SEQ ID NO: 106), LCDR2 (SEQ ID NO: 117), and LCDR3 (SEQ ID NO: 128); Ab2 LCv CDR: LCDR1 (SEQ ID NO: 107), LCDR2 (SEQ ID NO: 118) and LCDR3 (SEQ ID NO: 129); CDRs of Ab3 LCv: LCDR1 (SEQ ID NO: 108), LCDR2 (SEQ ID NO: 119), and LCDR3 (SEQ ID NO: 130); CDRs of Ab4 LCv: LCDR1 (SEQ ID NO: 109), LCDR2 (SEQ ID NO: 120) and LCDR3 (SEQ ID NO: 131); CDRs of Ab5 LCv: LCDR1 (SEQ ID NO: 110), LCDR2 (SEQ ID NO: 121) and LCDR3 (SEQ ID NO: 132); CDRs of Ab6 LCv: LCDR1 (SEQ ID NO: 111), LCDR2 (SEQ ID NO: 122) and LCDR3 (SEQ ID NO: 133); CDR of Ab7 LCv : LCDR1 (SEQ ID NO: 112), LCDR2 (SEQ ID NO: 123), and LCDR3 (SEQ ID NO: 134); CDRs of Ab8 LCv: LCDR1 (SEQ ID NO: 113), LCDR2 (SEQ ID NO: 124) And LCDR3 (SEQ ID NO: 135); CDRs of Ab9 LCv: LCDR1 (SEQ ID NO: 114), LCDR2 (SEQ ID NO: 125), and LCDR3 (SEQ ID NO: 136); Ab10 LCv CDR: LCDR1 (SEQ ID NO: 115), LCDR2 (SEQ ID NO: 126) and LCDR3 (SEQ ID NO: 137); CDRs of Ab11 LCv: LCDR1 (SEQ ID NO: 116), LCDR2 (SEQ ID NO: 127), and LCDR3 (SEQ ID NO: 138); CDRs of Ab30 LCv: LCDR1 (SEQ ID NO: 166), LCDR2 (SEQ ID NO: 169) and LCDR3 (SEQ ID NO: 172); Ab32 LCv CDR: LCDR1 (SEQ ID NO: 167 ), LCDR2 (SEQ ID NO: 170) and LCDR3 (SEQ ID NO: 173); Ab33 LCv CDRs: LCDR1 (SEQ ID NO: 168), LCDR2 (SEQ ID NO: 171), and LCDR3 (SEQ ID NO: 1) 74); CDRs of Ab1 HCv: HCDR1 (SEQ ID NO: 40), HCDR2 (SEQ ID NO: 51) and HCDR3 (SEQ ID NO: 62); CDRs of Ab2 HCv: HCDR1 (SEQ ID NO: 41), HCDR2 (SEQ ID NO: 52) and HCDR3 (SEQ ID NO: 63); CDRs of Ab3 HCv: HCDR1 (SEQ ID NO: 42), HCDR2 (SEQ ID NO: 53) and HCDR3 (SEQ ID NO: 64); CDRs of Ab4 HCv: HCDR1 (SEQ ID NO: 43), HCDR2 (SEQ ID NO: 54) and HCDR3 (SEQ ID NO: 65); CDRs of Ab5 HCv: HCDR1 (SEQ ID NO: 44), HCDR2 (SEQ ID NO: 55) and HCDR3 (SEQ ID NO: 66); CDRs of Ab6 HCv : HCDR1 (SEQ ID NO: 45), HCDR2 (SEQ ID NO: 56) and HCDR3 (SEQ ID NO: 67); CDRs of Ab7 HCv: HCDR1 (SEQ ID NO: 46), HCDR2 (SEQ ID NO: 57) And HCDR3 (SEQ ID NO: 68); CDRs of Ab8 HCv: HCDR1 (SEQ ID NO: 47), HCDR2 (SEQ ID NO: 58) and HCDR3 (SEQ ID NO: 69); CDRs of Ab9 HCv: HCDR1 (SEQ ID NO: 48), HCDR2 (SEQ ID NO: 59) and HCDR3 (SEQ ID NO: 70); CDRs of Ab10 HCv: HCDR1 (SEQ ID NO: 49), HCDR2 (SEQ ID NO: 60) and HCDR3 (SEQ ID NO: 71); CDRs of Ab11 HCv: HCDR1 (SEQ ID NO: 50), HCDR2 (SEQ ID NO: 61) and HCDR3 (SEQ ID NO: 72); Ab30 HCv CDR: HCDR1 (SEQ ID NO: 148 , HCDR2 (SEQ ID NO: 151) and HCDR3 (SEQ ID NO: 154); CDRs of Ab32 HCv: HCDR1 (SEQ ID NO: 149), HCDR2 (SEQ ID NO: 152) and HCDR3 (SEQ ID NO: 155) ); and Ab33 HCv CDR: HC DR1 (SEQ ID NO: 150), HCDR2 (SEQ ID NO: 153) and HCDR3 (SEQ ID NO: 156).

In some embodiments, the antigen binding protein comprises: A) a polypeptide, eg, a light chain, comprising an LCDR1 having an amino acid sequence selected from the group consisting of: SEQ ID NO: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 166, 167 and 168; LCDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 169, 170 and 171; and/or LCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 172, 173, and 174; and/or B) a polypeptide, eg, a heavy chain, comprising a population selected from the group consisting of HCDR1 of the amino acid sequence: SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 148, 149 and 150; having an amino acid selected from the group consisting of a sequence of HCDR2: SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 151, 152, and 153; and/or having an amino acid selected from the group consisting of Sequence HCDR3: SEQ ID NO: 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 154, 155 and 156.

In other embodiments, the antigen binding protein comprises A) a light chain amino acid sequence comprising: Ab1 LCv, Ab2 LCv, Ab3 LCv, Ab4 LCv, Ab5 LCv, Ab6 LCv, Ab7 LCv, Ab8 LCv, Ab9 LCv, Ab10 LCDR1, LCDR2 and LCDR3 of any of LCv, Ab11 LCv, Ab30 LCv, Ab32 LCv and Ab33 LCv; and B) heavy amino acid sequence comprising: Ab1 HCv, Ab2 HCv, Ab3 HCv, Ab4 HCv, Ab5 HCDR1, HCDR2 and HCDR3 of any of HCv, Ab6 HCv, Ab7 HCv, Ab8 HCv, Ab9 HCv, Ab10 HCv, Ab11 HCv, Ab30 HCv, Ab32 HCv and Ab33 HCv.

In certain embodiments, the CDRs differ from the exemplary CDRs described herein by no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, or no more than 6 amino acid additions. , missing or replaced.

The present invention includes an antibody comprising a light chain variable domain selected from the group consisting of SEQ ID NO: 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 163, 164 and 165. The present invention includes an antibody comprising a heavy chain variable domain selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 145, 146 and 147. Other aspects of the invention include antibodies comprising: A) a light chain variable domain selected from the group consisting of SEQ ID NOs: 95, 96, 97, 98, 99, 100, 101, 102, 103, 104 , 105, 163, 164 and 165, and B) a heavy chain variable domain selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 145, 146 and 147.

The antibodies of the invention may comprise any constant region known in the art. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, such as a human kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma- or mu-type heavy chain constant region, such as a human alpha-, delta-, eps-, gamma-, or mu-type heavy chain constant Area. In one embodiment, the light or heavy chain constant region is a fragment, derivative, variant or mutant protein in which a constant region is naturally present.

The invention includes an antibody comprising a light chain variable region selected from the group consisting of SEQ ID NO: 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 163, 164 And 165, the sequences having no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9 Adding, deleting or substituting no more than 10 amino acids. The present invention includes an antibody comprising a heavy chain variable region selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 145, 146. And 147, the sequences having no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9 Adding, deleting or substituting no more than 10 amino acids. Other aspects of the invention include antibodies comprising: A) a light chain variable region selected from the group consisting of: SEQ ID NO: 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 163, 164 and 165, the sequences having no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no More than 6, no more than 7, no more than 8, no more than 10 or no more than 10 amino acid additions, deletions or substitutions; and B) a heavy chain variable region selected from the group consisting of: SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 145, 146 and 147, the sequences have no more than 1, no more than 2, no more than 3, no More than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9 or no more than 10 amino acid additions, deletions or substitutions.

In a variation, the antigen binding protein comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85% of the light chain variable region amino acid sequence selected from the group consisting of At least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical amino acid sequence: SEQ ID NO: 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 163, 164 and 165. In another variation, the antigen binding protein comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85 of a heavy chain variable region amino acid sequence selected from the group consisting of %, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, At least 98% or at least 99% identical amino acid sequence: SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 145, 146 and 147. In another embodiment, the antigen binding protein comprises A) at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, and a light chain variable region amino acid sequence selected from the group consisting of At least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98% or at least 99% identical amino acid sequence: SEQ ID NO: 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 163, 164 and 165; And at least 80%, at least 81%, at least 82%, to a heavy chain variable region amino acid sequence selected from the group consisting of 83% less, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95 %, at least 96%, at least 97%, at least 98% or at least 99% identical amino acid sequence: SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 145, 146 and 147.

In certain embodiments, the antigen binding protein comprises a light chain and/or a heavy chain CDR3. In some embodiments, the antigen binding protein comprises a plurality selected from the group consisting of SEQ ID NO: 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 172, 173, 174, 62, 63, 64, The amino acid sequence of the group of sequences described in 65, 66, 67, 68, 69, 70, 71, 72, 154, 155 and 156. In certain embodiments, the amino acid sequence is SEQ ID NO: 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 172, 173, 174, 62, 63, 64, The example sequences described in 65, 66, 67, 68, 69, 70, 71, 72, 154, 155, and 156 differ by no more than 1, no more than 2, no more than 3, no more than 4, no more than Addition, deletion or substitution of 5 or no more than 6 amino acids. Accordingly, embodiments of the invention include and are selected from the group consisting of SEQ ID NO: 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 172, 173, 174, 62, 63, 64, 65 At least 80%, at least 81%, at least 82%, at least 83%, at least 84% of the amino acid sequence of the sequences of 66, 67, 68, 69, 70, 71, 72, 154, 155 and 156 At least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least An antigen binding protein of 97%, at least 98% or at least 99% identical amino acid sequence.

In certain embodiments, the antigen binding protein comprises a light chain and/or a heavy chain CDR2. In some embodiments, the antigen binding protein comprises a plurality selected from the group consisting of SEQ ID NO: 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 169, 170, 171, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 151, 152 and 153 Amino acid sequence of the population of said sequences. In certain embodiments, the amino acid sequence is SEQ ID NO: 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 169, 170, 171, 51, 52, 53, The example sequences described in 54, 55, 56, 57, 58, 59, 60, 61, 151, 152, and 153 differ by no more than 1, no more than 2, no more than 3, no more than 4, no more than Addition, deletion or substitution of 5 or no more than 6 amino acids. Accordingly, embodiments of the invention include and are selected from the group consisting of SEQ ID NO: 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 169, 170, 171, 51, 52, 53, 54 At least 80%, at least 81%, at least 82%, at least 83%, at least 84% of the amino acid sequence of the sequences of 55, 56, 57, 58, 59, 60, 61, 151, 152 and 153 At least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least An antigen binding protein of 97%, at least 98% or at least 99% identical amino acid sequence.

In certain embodiments, the antigen binding protein comprises a light chain and/or a heavy chain CDR1. In some embodiments, the antigen binding protein comprises a plurality selected from the group consisting of SEQ ID NO: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 166, 167, 168, 40, 41, 42, The amino acid sequence of the group of sequences described in 43, 44, 45, 46, 47, 48, 49, 50, 148, 149 and 150. In certain embodiments, the amino acid sequence is SEQ ID NO: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 166, 167, 168, 40, 41, 42, The example sequences described in 43, 44, 45, 46, 47, 48, 49, 50, 148, 149 and 150 differ by no more than 1, no more than 2, no more than 3, no more than 4, no more than Addition, deletion or substitution of 5 or no more than 6 amino acids. Accordingly, embodiments of the invention include and are selected from the group consisting of SEQ ID NO: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 166, 167, 168, 40, 41, 42, 43 At least 80%, at least 81%, at least 82%, at least the amino acid sequence of the sequences of 44, 45, 46, 47, 48, 49, 50, 148, 149 and 150 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% An antigen binding protein of at least 96%, at least 97%, at least 98%, or at least 99% of an amino acid sequence that is identical.

The antigen-binding protein of the present invention comprises a scaffold of a conventional antibody, including human and monoclonal antibodies, bispecific antibodies, bifunctional antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as " Antibody mimics"), chimeric antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and fragments thereof, respectively. The above CDRs (including various combinations of CDRs) can be grafted into any of the following scaffolds.

The term "antibody" as used herein, refers to a monomer or multimeric protein comprising one or more polypeptide chains that specifically bind to an antigen, as described herein in a different manner. In certain embodiments, the anti-system is produced by recombinant DNA techniques. In other embodiments, the anti-system is produced by enzymatic or chemical cleavage of a naturally occurring antibody. In another aspect, the anti-system is selected from the group consisting of: a) human antibodies, b) humanized antibodies, c) chimeric antibodies, d) monoclonal antibodies, e) polyclonal antibodies, f) recombinant antibodies, g) antigen-binding fragment, h) single chain antibody, i) diabody, j) diabody, k) diabody, 1) Fab fragment, m) F(ab') ₂ fragment, n) IgA antibody o) IgD antibody, p) IgE antibody, q) IgG1 antibody, r) IgG2 antibody, s) IgG3 antibody, t) IgG4 antibody, and u) IgM antibody.

The variable region or domain comprises at least 3 heavy or light chain CDRs within the framework regions (designated frame regions FR1, FR2, FR3 and FR4). Kabat et al., 1991, Sequences of Proteins of Immunological Interest , Public Health Service NIH, Bethesda, MD. Traditional antibody structural units typically comprise a tetramer. Each tetramer is typically composed of two identical polypeptide chain pairs, each pair having a "light" chain and a "heavy" chain. The amino terminus portion of each chain includes a variable region having from about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy terminal portion of each chain defines a constant region that is primarily responsible for effector function. Human light chains are classified as kappa or lambda light chains. The heavy chain is classified as μ, δ, γ, α or ε, and the isotypes of the antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subclasses including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. Embodiments of the invention include all such classes and subclasses of antibodies that incorporate the variable domains or CDRs of the antigen binding proteins described herein.

Some naturally occurring antibodies (such as those found in camels and camel horses) are dimers composed of two heavy chains and do not include light chains. The invention encompasses dimeric antibodies or fragments thereof that bind to the two heavy chains of ST2.

The variable regions of the heavy and light chains typically exhibit the same general structure of the relatively conserved framework regions (FR) joined by three hypervariable regions (i.e., complementarity determining regions or CDRs). The CDR is primarily responsible for antigen recognition and binding. The CDRs from the two strands of each pair are aligned by the framework regions, enabling them to bind to specific epitopes. From the N-terminus to the C-terminus, both the light chain and the heavy chain comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids in each domain is in accordance with the definition of Kabat.

The CDRs constitute the major surface contact point for antigen binding. The CDR3 of the light chain and especially the CDR3 of the heavy chain can constitute the most important determinant of antigen binding in the light chain and heavy chain variable regions. In some antibodies, the heavy chain CDR3 appears to constitute the primary contact area between the antigen and the antibody. An in vitro selection scheme that only alters CDR3 can be used to alter the binding properties of the antibody or to determine residues that contribute to antigen binding.

Naturally occurring antibodies typically include a signal sequence that directs the antibody to a cellular pathway for protein secretion and is typically not present in the mature antibody. A polynucleotide encoding an antibody of the invention may encode a naturally occurring signal sequence or a heterologous signal sequence, as described below.

In one embodiment, the antigen binding protein line comprises from 1 to 6 antibodies of the exemplary CDRs described herein. The antibodies of the invention may be of any type, including IgM, IgG (including IgGl, IgG2, IgG3, IgG4), IgD, IgA or IgE antibodies. In a particular embodiment, the antigen binding protein is an IgG type antibody, eg, an IgGl antibody.

In some embodiments, for example, when the antigen binding protein line has a complete heavy chain and In the case of antibodies to the light chain, the CDRs are all from the same species, for example, humans. Alternatively, for example, in embodiments where the antigen binding protein contains less than 6 CDRs from the sequences outlined above, the other CDRs may be from other species or may be different from the human CDRs depicted in the exemplary sequences. For example, HCDR3 and LCDR3 regions from suitable sequences identified herein can be used with HCDR1, HCDR2, LCDR1, and LCDR2, optionally selected from alternative species or different human antibody sequences, or a combination thereof. For example, a CDR of the invention can be substituted for a CDR region of a commercially relevant chimeric or humanized antibody.

A specific embodiment utilizes an antigen binding protein as a scaffold component of a human component. However, in some embodiments, the scaffold component can be a mixture from different species. Thus, if the antigen binds to a protein-based antibody, the antibody can be a chimeric antibody and/or a humanized antibody. In general, both "chimeric antibody" and humanized antibody" refer to antibodies that combine regions from more than one species. For example, a "chimeric antibody" typically comprises a variable region from a mouse (or rat, in some cases) and a constant region from a human.

"Humanized antibody" generally refers to a non-human antibody in which the variable domain framework region has been exchanged for a sequence found in a human antibody. Typically, in a humanized antibody, the entire antibody (except for one or more CDRs) is encoded by a human-derived polynucleotide or is identical to such an antibody except within one or more CDRs. Some or all of the CDRs encoded by the nucleic acid derived from the non-human organism are grafted into the beta sheet of the variable region of the human antibody to produce an antibody that is specifically determined by the CDRs. The production of such antibodies is described, for example, in WO 92/11018; Jones 1986, Nature 321:522-525; Verhoeyen et al, 1988, Science 239: 1534-1536. Humanized antibodies can also be generated using mice with a genetically engineered immune system. Roquc et al., 2004, Biotechnol. Prog. 20:639-654. In the exemplary embodiments described herein, the identified CDRs are human, and thus, both humanized and chimeric antibodies in this context include some non-human CDRs; for example, HCDR3 and LCDR3 regions can be generated and others One or more of the CDR regions have humanized antibodies of different species origin.

In one embodiment, the ST2 antigen binding protein is a multispecific antibody, and in particular is a bispecific antibody, sometimes referred to as a "bifunctional antibody." These are antibodies that bind to two or more different antigens or different epitopes on a single antigen. In certain embodiments, the bispecific antibody binds to an antigen on ST2 and human effector cells (eg, T cells). Such antibodies can be used to target effector cell responses to cells expressing ST2, such as tumor cells that express ST2. In a preferred embodiment, the human effector cell antigen line is CD3. U.S. Patent No. 7,235,641. Methods of making bispecific antibodies are known in the art. One such method involves engineering the Fc portion of the heavy chain to create "knobs" and "holes" which, when co-expressed in the cell, contribute to the formation of heterodimers in the heavy chain. U.S. 7,695,963. Another approach also involves engineering the Fc portion of the heavy chain, but using electrostatic traction to promote heterodimer formation when co-presented in the cell while preventing the heavy chain from forming a homodimer. WO 09/089,004, the entire contents of which is incorporated herein by reference.

In one embodiment, the ST2 antigen binds to a protein line minibody. The mini-antibody system comprises a minimal antibody-like protein that scFv binds to the CH3 domain. Hu et al., 1996, Cancer Res. 56: 3055-3061.

In one embodiment, the ST2 antigen binds to a protein domain domain antibody; see, for example, U.S. Patent No. 6,248,516. A domain antibody (dAb) is a functional binding domain of an antibody that corresponds to the variable region of the heavy (VH) strand or light (VL) strand of a human antibody. The dAB has a molecular weight of about 13 kDa or less than 1/10 of the size of the full antibody. dAB is fully expressed in a variety of hosts, including bacterial, fungal, and mammalian cell systems. In addition, the dAb is highly stable and retains activity even after being subjected to harsh conditions such as cold-drop drying or heat denaturation. See, for example, U.S. Patent Nos. 6,291,158, 6, 582, 915, 6, 593, 081, 6, 172, 197; U.S. Patent No. 2004/0110941; European Patent No. 0368684; U.S. Patent No. 6,696,245; WO04/058821; WO04/003019 and WO03/002609.

In one embodiment, the ST2 antigen binds to a protein-based antibody fragment, that is, a fragment of any of the antibodies that retain the binding specificity for ST2 as outlined herein. In various embodiments, resistant Body-binding proteins include, but are not limited to, F(ab), F(ab'), F(ab')2, Fv or single-chain Fv fragments. Minimally, an antibody as referred to herein comprises a polypeptide that specifically binds to ST2, which comprises all or a portion of a light or heavy chain variable region, such as one or more CDRs.

Other examples of antibody fragments that bind to ST2 include, but are not limited to, (i) a Fab fragment consisting of VL, VH, CL, and CH1 domains; (ii) an Fd fragment consisting of VH and CH1 domains; Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment (Ward et al., 1989, Nature 341:544-546) consisting of a single variable domain; (v) isolated CDR region; (vi) F(ab') ₂ fragment, a bivalent fragment comprising two linked Fab fragments; (vii) a single-chain Fv molecule (scFv), wherein the VH domain and the VL domain are joined by a peptide A ligation that allows two domains to associate to form an antigen binding site (Bird et al, 1988, Science 242: 423-426; Huston et al, 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883); (viii) bispecific single-chain Fv dimer (PCT/US92/09965); and (ix) "bifunctional antibody" or "trifunctional antibody", multivalent constructed by gene fusion or Multispecific fragments (Tomlinson et al, 2000, Methods Enzymol. 326:461-479; WO 94/13804; Holliger et al, 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448). Antibody fragments can be modified. For example, such molecules can be stabilized by the inclusion of a disulfide bridge linking the VH and VL domains (Reiter et al., 1996, Nature Biotech. 14: 1239-1245). Aspects of the invention include embodiments in which the non-CDR components of the fragments are human sequences.

In one embodiment, the ST2 antigen binding protein is a fully human antibody. In this embodiment, as outlined above, the specific structure comprises the complete heavy and light chains comprising the CDR regions as described. Other embodiments utilize one or more CDRs of the invention with other CDRs, framework regions, J and D regions, constant regions, and the like from other human antibodies. For example, a CDR of the invention can be substituted for the CDRs of any number of human antibodies, particularly commercially relevant antibodies.

Single-chain antibodies can be formed by ligating a heavy chain to a light chain variable domain (Fv region) fragment with an amino acid bridge (short peptide linker) to produce a single polypeptide chain. These single-chain Fv (scFv) can already by encoding the fusion peptide linker DNA between the DNA of variable domain polypeptide (V _L and V _H) are prepared encoding two. The resulting polypeptide can be folded back by itself to form an antigen-binding monomer, or it can form a polymer (eg, a dimer, a trimer, or a tetramer), which is a flexible link between the two variable domains. Depending on the length of the body (Kortt et al, 1997, Prot. Eng. 10: 423; Kortt et al, 2001, Biomol. Eng. 18: 95-108). By containing different combinations of V _L and V _H of the polypeptide, bind to different epitopes can be formed as much as poly scFv (Kriangkum et al., 2001, Biomol.Eng.18: 31-40). Techniques developed for the production of single-chain antibodies include those described in U.S. Patent No. 4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879; Ward et al, 1989, Nature 334: 544; de Graaf et al, 2002, Methods Mol Biol. 178: 379-87. The invention encompasses single chain antibodies derived from the antibodies provided herein, including, but not limited to, scFv comprising: a combination of the following variable domains: Ab1 LCv/Ab1 HCv (SEQ ID NO: 95/SEQ ID NO: 29), Ab2 LCv/Ab2 HCv (SEQ ID NO: 96/SEQ ID NO: 30), Ab3 LCv/Ab3 HCv (SEQ ID NO: 97/SEQ ID NO: 31), Ab4 LCv/Ab4 HCv (SEQ ID NO: 98/ SEQ ID NO: 32), Ab5 LCv/Ab5 HCv (SEQ ID NO: 99/SEQ ID NO: 33), Ab6 LCv/Ab6 HCv (SEQ ID NO: 100/SEQ ID NO: 34), Ab7 LCv/Ab7 HCv (SEQ ID NO: 101 / SEQ ID NO: 35), Ab8 LCv/Ab8 HCv (SEQ ID NO: 102 / SEQ ID NO: 36), Ab9 LCv/Ab9 HCv (SEQ ID NO: 103 / SEQ ID NO: 37 ), Ab10 LCv/Ab10 HCv (SEQ ID NO: 104/SEQ ID NO: 38) and Ab11 LCv/Ab11 HCv (SEQ ID NO: 105/SEQ ID NO: 39), Ab30 LCv/Ab30 HCv (SEQ ID NO: 163/SEQ ID NO: 145), Ab32 LCv/Ab32 HCv (SEQ ID NO: 164/SEQ ID NO: 146), Ab33 LCv/Ab33 HCv (SEQ ID NO: 165/SEQ ID NO: 147), and combinations thereof.

In one embodiment, the ST2 antigen binds to a protein-based antibody fusion protein (sometimes referred to herein as an "antibody conjugate"). The conjugate object can be proteinaceous or non-protein Qualitative; the latter is typically produced using antigen-binding proteins and functional groups on conjugate targets. In certain embodiments, the antibody is coupled to a non-proteinaceous chemical (drug) to form an antibody drug conjugate.

In one embodiment, the ST2 antigen binding protein line antibody analog is sometimes referred to as a "synthetic antibody." For example, a variety of products utilize alternative protein scaffolds or artificial scaffolds with grafted CDRs. Such scaffolds include, but are not limited to, mutations introduced to stabilize the three-dimensional structure of the bound protein and fully synthetic scaffolds composed of, for example, biocompatible polymers. See, for example, Korndorfer et al, 2003, Proteins: Structure, Function, and Bioinformatics , Vol. 53, No. 1: 121-129; Roque et al, 2004, Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics ("PAM"), as well as antibody mimetic based products, in which the fibronectin linker component is utilized as a scaffold, can be used.

As used herein, "protein" means at least two covalently attached amino acids including proteins, polypeptides, oligopeptides, and peptides. In some embodiments, two or more covalently attached amino acids are attached by peptide bonds. For example, when a protein is produced using recombinant expression systems and host cell recombination as outlined below, the protein may be composed of naturally occurring amino acids and peptide bonds. Alternatively, the protein may comprise a synthetic amino acid (eg, high amphetamine, citrulline, ornithine, and orthanoic acid) or a peptidomimetic structure that is resistant to proteases or other physiological and/or storage conditions, ie, "Peptide or protein analog", such as a peptoid (see, Simon et al, 1992, Proc. Natl. Acad. Sci. USA 89:9367, herein incorporated by reference). Such synthetic amino acids are especially useful when the antigen-binding proteins are synthesized in vitro by conventional methods well known in the art. In addition, any combination of peptidomimetics, synthetic and naturally occurring residues/structures can be used. "Amino acids" also include imido acid residues such as valine and hydroxyproline. The amino acid "R group" or "side chain" may be in the (L)- or (S)-configuration. In a particular embodiment, the amino acid is in the (L)- or (S)-configuration.

In some aspects, the invention provides for the binding of ST2 and, in some embodiments, recombinant humans Recombinant antigen binding protein of ST2 or a portion thereof. In this context, "recombinant protein" is a protein produced using recombinant techniques using any of the techniques and methods known in the art (i.e., by the performance of recombinant nucleic acids as described herein). Methods and techniques for producing recombinant proteins are well known in the art. Embodiments of the invention include recombinant antigen binding proteins that bind wild type ST2 and variants thereof.

"Consisting essentially of" means that the amino acid sequence can be altered by about 1%, 2%, 3%, 4%, 5%, 6%, 7 relative to the sequence recited as SEQ ID NO: %, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% and still retain biological activity, as described herein.

In some embodiments, the antigen binding proteins of the invention are isolated proteins or substantially pure proteins. "Separated" proteins are not accompanied by at least some of the materials normally associated with the natural state, for example, constituting at least about 5% by weight or at least about 50% by weight of the total protein in a given sample. It will be appreciated that depending on the environment, the isolated protein may constitute from 5% to 99.9% by weight of the total protein content. For example, the protein can be made at a significantly higher concentration by using an inducible promoter or a high performance promoter such that the protein is made in an increased concentration. This definition includes the production of antigen binding proteins in a wide variety of organisms and/or host cells known in the art.

For amino acid sequences, sequence identity and/or similarity is determined by using standard techniques known in the art including, but not limited to, those of Smith and Waterman, 1981, Adv. Appl. Math. 2:482. Sequence Consistency Algorithm, Needleman and Wunsch, 1970, J. Mol. Biol. 48:443 Sequence Consistency Alignment Algorithm, Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444 Sex search method, computerized implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), by Devereux et al., 1984, Nucl.Acid Res. 12:387-395 illustrates the best fit sequence program, preferably using preset settings or by detection. Preferably, the percent consistency is calculated by FastDB based on the following parameters: a mismatch penalty of 1; a gap penalty of 1; a gap penalty of 0.33; and a junction penalty of 30, "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss.

An example of an available algorithm is PILEUP. PILEUP uses a progressive contrast pair to generate multiple sequence alignments from a set of related sequences. It can also draw a tree that is used to generate a clustered display of the alignment. PILEUP uses a simplified version of the progressive alignment method of Feng and Doolittle, 1987, J. Mol. Evol. 35:351-360; this method is similar to that described by Higgins and Sharp, 1989, CABIOS 5: 151-153. The available PILEUP parameters include a preset gap weight of 3.00, a preset gap length weight of 0.10, and a weighted end slot.

Another example of an available algorithm is described in the BLAST algorithm in "Altschul et al., 1990, J. Mol. Biol. 215: 403-410; Altschul et al., 1997, Nucleic Acids Res. 25:3389- 3402; and Karin et al., 1993, Proc. Natl. Acad. Sci. USA 90:5873-5787. The BLAST program is particularly useful as WU-BLAST available from Altschul et al., 1996, Methods in Enzymology 266:460-480. -2 program. WU-BLAST-2 uses several search parameters, most of which are set to preset values. The adjustable parameters are set to the following values: overlap span = 1, overlap score = 0.125, word threshold (T) = II. The HSP S and HSP S2 parameters are dynamic values and are determined by the program itself, depending on the composition of the particular sequence and the composition of the particular database being searched for the sequence of interest; however, the values can be adjusted to increase sensitivity.

Another available algorithm is vacancy BLAST, as reported by Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402. Vacancy BLAST uses BLOSUM-62 instead of scoring; threshold T parameter is set to 9; use two-hit method to trigger no-vacancy extension; requires k vacancy length to have a cost of 10+k; X _{u is} set to 16; and X _{g is} set to 40 in the database search phase and 67 in the output phase of the algorithm. A vacancy alignment is triggered corresponding to a score of approximately 22 bits.

Typically, the amino acid homology, similarity or identity between the individual variant CDRs and the sequences depicted herein is at least 80%, and more typically has at least 85%, 90%, 91%, 92%. , 93%, 94%, 95%, 96%, 97%, 98%, 99%, and almost 100% increase in homology or consistency. In a similar manner, the "percent sequence identity (%) of nucleic acid sequence" for the nucleic acid sequence of the binding protein identified herein is defined as the nucleotide residue in the candidate sequence that is identical to the nucleotide residue in the coding sequence of the antigen-binding protein. Percentage. The specific method utilizes the BLASTN module of WU-BLAST-2 set as a preset parameter, in which the overlap span and the overlap score are set to 1 and 0.125, respectively.

Typically, the nucleic acid sequence homology, similarity or identity between the nucleotide sequence encoding the individual variant CDRs and the nucleotide sequence depicted herein is at least 80%, and more typically preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98% or 99% and almost 100% increase in homology or consistency.

Thus, a "variant CDR" has a specified homology, similarity or identity to a parent CDR of the invention and shares at least the biological function, including but not limited to the specificity and/or activity of the parent CDR. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98% or 99%.

Although the site or region for introducing the amino acid sequence variation is predetermined, the mutation itself need not be predetermined. For example, to optimize the performance of a mutation at a localization site, random mutagenesis can be performed at the target codon or target region and the optimal combination of the desired activities indicative of the CDR variant of the antigen binding protein can be screened. Techniques for achieving substitution mutations at predetermined positions in DNA having known sequences are well known in the art, for example, M13 primer mutagenesis and PCR mutagenesis. Screening of mutants is performed using antigen binding protein activity assays (eg, ST2 binding).

The amino acid substitution is typically a single residue; the insertion will typically be from about one (1) to about twenty (20) amino acid residues, but a significantly larger insertion can be tolerated. The deletion is in the range of from about one (1) to about twenty (20) amino acid residues, but in some cases the deletion can be much larger.

Substitutions, deletions, insertions, or any combination thereof can be used to achieve the final derivative or variant. Typically, these changes are made to several amino acids to minimize the alteration of the molecule, particularly the immunogenicity and specificity of the antigen binding protein. However, in some cases larger changes can be tolerated. Conservative substitutions are usually carried out according to the table as depicted in Table 3.

Substantial changes in functional or immunological properties are less conservative than by Table 3 The replacement is implemented. For example, substitutions that more significantly affect the structure of the polypeptide backbone in the region, such as an alpha helix or beta sheet structure; the charge or hydrophobicity of the molecule at the target site; or the size of the side chain, can be implemented. In general, substitutions that are expected to produce the greatest change in the nature of the polypeptide are those in which (a) a hydrophilic residue (eg, a serine or a sulfinyl group) is (or consists of) a hydrophobic residue (eg, a bright amine) a thiol, isoleucido, amphetamine, amidino or propylamine amide; (b) a cysteine or valine acid substituted with (or by) any other residue; c) a residue having a positively charged side chain (for example, an amine sulfhydryl group, a spermine sulfhydryl group or a histamine sulfhydryl group) via (or by) a negatively charged residue (for example, glutamine sulfhydryl or aspartame) a substituent; or (d) a residue having a bulky side chain (eg, phenylalanine) substituted with (or by) a moiety that does not have a side chain (eg, glycine).

Variants typically exhibit the same qualitative biological activity as a naturally occurring analog and will elicit the same immune response as the analog, but variants are also selected as needed to modify the properties of the antigen binding protein. Alternatively, the variant can be designed to alter the biological activity of the antigen binding protein. For example, a glycosylation site can be altered or removed as discussed herein.

Other derivatives of ST2 antibodies within the scope of the invention include covalent or aggregated conjugates of ST2 antibodies or fragments thereof with other proteins or polypeptides, for example by expression comprising a heterologous polypeptide and a ST2 antibody polypeptide N-terminus or C The end-fused recombinant fusion protein was obtained. For example, the coupled peptide can be a heterologous signal (or leader) polypeptide such as a yeast alpha factor leader, or a peptide such as an epitope tag. A fusion protein comprising an ST2 antibody can comprise a peptide (e.g., polyHis) added to facilitate purification or identification of the ST2 antibody. The ST2 antibody polypeptide can also be linked to a FLAG peptide as described in Hopp et al., Bio/Technology 6: 1204, 1988 and U.S. Patent 5,011,912. The FLAG peptide is highly antigenic and provides an epitope that reversibly binds to a specific monoclonal antibody (mAb), thereby enabling rapid analysis and convenient purification of the expressed recombinant protein. Reagents useful in the preparation of fusion proteins that fuse FLAG peptides to a given polypeptide are commercially available (Sigma, St. Louis, MO).

In one embodiment, a polypeptide derived from an immunoglobulin is used to prepare an oligomer. The preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of the antibody-derived polypeptide, including the Fc domain, has been described, for example, in Ashkenazi et al., 1991, PNAS USA 88: 10535; Byrn et al. Human, 1990, Nature 344: 677; and Hollenbaugh et al., 1992 "Construction of Immunoglobulin Fusion Proteins", Current Protocols in Immunology , Supplement 4, pages 10.19.1-10.19.11.

One embodiment of the invention pertains to a dimer comprising two fusion proteins produced by fusing an ST2 binding fragment of an ST2 antibody with an Fc region of an antibody. The dimer can be prepared, for example, by inserting a fusion gene encoding a fusion protein into a suitable expression vector, expressing the fusion of the gene in a host cell transformed with the recombinant expression vector, and making it very similar to the antibody. The fusion protein is expressed in a molecular manner, and then an interchain disulfide bond is formed between the Fc moieties to produce a dimer.

The term "Fc polypeptide" as used herein, includes both natural and mutant protein forms of a polypeptide derived from the Fc region of an antibody. Also included are truncated forms of the hinge regions of these polypeptides that promote dimerization. The fusion protein comprising the Fc portion (and the oligomer formed therefrom) provides the advantage of being readily purified by affinity chromatography on Protein A or Protein G columns.

A suitable Fc polypeptide that is described in PCT application WO 93/10151 (incorporated herein by reference) is a single-chain polypeptide that extends from the N-terminal hinge region to the native C-terminus of the human IgG antibody Fc region. Another useful Fc polypeptide is the Fc mutant protein described in U.S. Patent No. 5,457,035 and Baum et al., 1994, EMBO J. 13:3992-4001. The amino acid sequence of this mutant protein is identical to the native Fc sequence shown in WO 93/10151 except that the amino acid 19 has changed from Leu to Ala, the amino acid 20 has changed from Leu to Glu, and the amino acid 22 has Changed from Gly to Ala. Mutant proteins exhibit reduced affinity for Fc receptors.

In other embodiments, the heavy chain and/or light chain variable portion of the ST2 antibody can be substituted with a variable portion of the antibody heavy and/or light chain.

Another method of preparing oligomeric ST2 antibody derivatives involves the use of an lysine zipper. The lysine zipper domain is a peptide that promotes the oligomerization of proteins in which these domains are found. The lysine zipper was originally identified in several DNA-binding proteins (Landschulz et al., 1988, Science 240: 1759) and has since been found to be present in a variety of different proteins. Peptides naturally occurring from the amide zipper system and derivatives thereof which are dimerized or trimerized are known. An example of an lyophilic acid zipper domain suitable for the production of a soluble oligomeric protein is set forth in PCT application WO 94/10308, and an lyophilic acid zipper derived from pulmonary surfactant protein D (SPD) is described in Hoppe et al., 1994. , FEBS Letters 344:191, which is incorporated herein by reference. The use of a modified leucine zipper allows for stable trimerization of the heterologous protein with which it is fused, as described in Fanslow et al., 1994, Semin. Immunol. 6:267-78. In one method, a recombinant fusion protein comprising an ST2 antibody fragment or derivative fused to a leucine zipper peptide is expressed in a suitable host cell, and the formed soluble oligomeric ST2 antibody fragment or derivative is recovered from the culture supernatant.

Covalent modifications of antigen binding proteins are included within the scope of the invention and are typically, but not always, performed in a post-translational manner. Covalently modifying several types of antigen-binding proteins into a molecule by reacting a specific amino acid residue of the antigen-binding protein with an organic derivatizing agent capable of reacting with a selected side chain or N- or C-terminal residue .

The cysteamine sulfhydryl residue is most typically reacted with an alpha-haloacetate (and corresponding amine) (e.g., chloroacetic acid or chloroacetamide) to provide a carboxymethyl or carboxy oxime aminomethyl derivative. Cysteine thiol residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidazolyl)propionic acid, chloroethyl sulfonyl phosphate, N-alkyl Maleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercaptobenzoate, 2-chloromercapto-4-nitrophenol Or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

The histamine sulfhydryl residue is derivatized by reaction with diethylpyrocarbonate (pH 5.5-7.0), so that the reagent is relatively specific for the histamine sulfhydryl side chain. It is also possible to use p-bromophenylhydrazine methyl bromide; preferably in 0.1 M sodium dimethyl citrate (pH 6.0).

The quaternary acid group and the amine terminal residue are reacted with succinic anhydride or other carboxylic anhydride. Use this Derivatization of the reagents has the effect of reversing the charge of the amino acid residues. Other suitable reagents for derivatization of alpha-containing amino acid residues include sulfhydryl esters such as methyl pyrimimine methyl ester; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzene sulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.

The spermine thiol residue is modified by reaction with one or several conventional reagents, particularly benzamidine formaldehyde, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues due to the high pK _a of the guanidine functional group need embodiment under basic conditions. In addition, the reagents can be reacted with a group of an amine acid and an ε-amine group of arginine.

Specific modifications of the tyramine sulfhydryl residue can be carried out with particular attention to the introduction of spectral labels into the tyramine sulfhydryl residue by reaction with an aromatic diazonium compound or tetranitromethane. Most commonly, N-acetylmercaptoimidazole and tetranitromethane are used, respectively, to form the O-acetyl tyramine base material and the 3-nitro derivative. The tyramine thiol residue is iodinated using ¹²⁵ I or ¹³¹ I to prepare a labeled protein for use in a radioimmunoassay, and the above chloramine T method is suitable.

The pendant carboxyl group (aspartame or glutamine) is bonded to carbodiimide (R'-N=C=N--R') (eg 1-cyclohexyl-3-(2-) Selective modification of morphyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azaindole-4,4-dimethylpentyl)carbodiimide, wherein R And R' is an alkyl group which is different depending on the case. In addition, the aspartic acid sulfhydryl group and the glutamic acid sulfhydryl residue are converted into aspartame and glutamine sulfhydryl residues by reaction with ammonium ions.

Derivatization with a bifunctional agent can be used to crosslink the antigen binding protein to a water insoluble carrier matrix or surface for use in a variety of methods. Commonly used crosslinking agents include, for example, 1,1-bis(disazoethyl)-2-phenylethane; glutaraldehyde; N-hydroxysuccinimide, for example, with 4-azido An ester of salicylic acid; a homobifunctional quinone imide, including a disuccinimide ester, such as 3,3'-dithiobis(piperidinyl propionate); and a bifunctional Malayan An amine such as bis-N-maleimido-1,8-octane. Derivatizing agents such as 3-[(p-azidophenyl)dithio]propionate methyl esters produce photoactivatable intermediates capable of forming crosslinks in the presence of light. Another option is to use reactive water-insoluble groups such as cyanogen bromide-activated carbohydrates. The reactive substrates described in U.S. Patent Nos. 3,969,287, 3,691,016, 4,195,128, 4,247,642, 4,229,537, and 4,330,440 are incorporated herein by reference.

The glutamic acid sulfhydryl group and the aspartame sulfhydryl residue are usually removed from the guanamine group to the corresponding glutamine sulfhydryl group and the aspartame group. Alternatively, the residues are removed from the guanamine group under mildly acidic conditions. Any of these residues is within the scope of the invention.

Other modifications include hydroxylation of lysine and lysine, phosphorylation of hydroxyl groups of serine sulfhydryl or sulphide residues, amides of lysine, arginine and histidine Methylation (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, 1983, pp. 79-86), acetylation of N-terminal amines and amide amination of any C-terminal carboxyl group .

Another type of covalent modification of an antigen binding protein encompassed within the scope of the invention involves altering the glycosylation pattern of the protein. As is known in the art, the glycosylation pattern can depend on the sequence of the protein (e.g., the presence or absence of a particular glycosylated amino acid residue, discussed below) or both the host cell or organism from which the protein is produced. Specific performance systems are discussed below.

Polypeptide glycosylation is typically via N-linkage or O-linkage. N-linkage refers to the attachment of a carbohydrate moiety to the side chain of an aspartate residue. The tripeptide sequence aspartame-X-serine and aspartamide-X-threonine (where X is any amino acid other than proline) is used for enzymatic attachment of carbohydrate moieties and The recognition sequence of the indole side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide can form a potential glycosylation site. O-linked glycosylation refers to a sugar (N-ethyl galactosamine, galactose or xylose) and a hydroxyl amino acid (most commonly serine or threonine, but 5-hydroxyl can also be used Attachment of proline or 5-hydroxy lysine.

The glycosylation site is typically conveniently added to the antibody by altering the amino acid sequence such that the antibody contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Alternatively, one or more serine or threonine residues or one or more filaments may be added to the starting sequence. Substitution of the amino acid or threonine residue (for O-linked glycosylation sites) to achieve this change. For convenience, the antigen-binding protein amino acid is preferably altered via changes in the DNA level, particularly by mutating the pre-base of the DNA encoding the target polypeptide to generate codons that will be translated into the desired amino acid. .

Another means of increasing the number of carbohydrate moieties on an antigen-binding protein is by chemical or enzymatic coupling of the glycoside to the protein. Such procedures are advantageous in that they do not require the production of proteins in host cells having the ability to glycosylate N- and O-linked glycosylation. Depending on the coupling mode used, the sugar may be attached to (a) arginine and histidine; (b) free carboxyl; (c) free sulfhydryl, such as cysteine; (d) free hydroxy , such as, for example, serine, threonine or hydroxyproline; (e) aromatic residues such as phenylalanine, tyrosine or tryptophan; or (f) glutamic acid Guanamine. Such methods are described in WO 87/05330, published September 11, 1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem. , pp . 259-306 .

The carbohydrate moiety present on the starting antigen binding protein can be removed chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment can lyse most or all of the sugar except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine) while leaving the intact polypeptide. Chemical deglycosylation is described in Hakimuddin et al, 1987, Arch. Biochem. Biophys. 259:52 and Edge et al, 1981, Anal. Biochem. 118:131. Enzymatic cleavage of the carbohydrate moiety on the polypeptide can be achieved by using a variety of endo- and exo-glycosidase enzymes as described in Thotakura et al., 1987, Meth. Enzymol. 138:350. Glycosylation at a potential glycosylation site can be prevented by the use of the compound tunicamycin as described in Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation of protein-N-glycosidic linkages.

Another type of covalent modification of an antigen binding protein comprises linking the antigen binding protein to various non-proteinaceous polymers in a manner described in the following patents including, but not limited to, various polyols (eg, polyethylene glycol, Polypropylene glycol) or polyoxyalkylene: American No. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 or 4,179,337. Additionally, as is known in the art, amino acid substitutions can be performed at various positions within the antigen binding protein to facilitate the addition of polymers such as PEG.

In some embodiments, covalent modification of an antigen binding protein of the invention comprises the addition of one or more labels.

The term "labelled group" means any detectable label. Examples of suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (eg, ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I) , fluorescent groups (eg, FITC, rhodamine, lanthanide phosphors), enzyme groups (eg, horseradish peroxidase, beta-galactosidase, luciferase, alkaline) a phosphatase), a chemiluminescent group, a biotin group, or a predetermined polypeptide epitope recognized by a secondary reporter (eg, a leucine zipper pair sequence, a secondary antibody binding site, a metal binding domain, an epitope) label). In some embodiments, the labeling group is coupled to the antigen binding protein via spacer arms of different lengths to reduce potential steric hindrance. Various methods of labeling proteins are known in the art and can be used to practice the invention.

In general, the assay at which the labeling end is to be detected is divided into a plurality of categories: a) isotope labeling, which may be radioactive or heavy isotopes; b) magnetic labeling (eg, magnetic particles); c) redox activity Part; d) optical dye; enzyme group (eg horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylated group; and f) by two A predetermined polypeptide epitope recognized by the reporter (eg, a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag, etc.). In some embodiments, the labeling group is coupled to the antigen binding protein via spacer arms of different lengths to reduce potential steric hindrance. Various methods of labeling proteins are known in the art and can be used to practice the invention.

Specific labels include optical dyes including, but not limited to, chromophores, phosphors, and fluorescent The group, the latter of which is specific in many cases. The fluorophore can be a "small molecule" fluore or a proteinaceous phosphor.

"Fluorescent label" means any molecule detectable by its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, luciferin, rose bengal, tetramethyl rose red, eosin, erythrosin, coumarin, methyl coumarin, anthraquinone, malachite green, alfalfa , Lucifer Yellow, Cascade BlueJ, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon Green ( Oregon green), Alexa-Fluor dye (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), waterfall blue, waterfall Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene, OR), FITC, Rose Bengal and Texas Red (Pierce, Rockford, IL), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh , PA). Suitable optical dyes, including chromophores, are described in Molecular Probes Handbook (Richard P. Haugland), which is expressly incorporated herein by reference.

Suitable proteinaceous fluorescent labels also include, but are not limited to, green fluorescent proteins, including GFP of Renilla, Ptilosarcus or Aequorea species (Chalfie et al, 1994, Science 263: 802- 805); EGFP (Clontech Laboratories, Genbank Accession No. U55762); Blue Fluorescent Protein (BFP, Quantum Biotechnologies, Inc., 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechnologies 24:462-471; Heim et al., 1996, Curr. Biol. 6: 178-182); Enhanced Yellow Fluorescent Protein (EYFP, Clontech Laboratories); Luciferase (Ichiki et al., 1993, J. Immunol) . 150: 5408-5417); β-galactosidase (Nolan et al., 1988, Proc.Natl.Acad.Sci.USA 85: 2603-2607) and Renilla (WO92 / 15673, WO95 / 07463 , WO98 / 14605 , WO 98/26277, WO 99/49019, U.S. Patent No. 5,292,658, No. 5,418,155, No. 5,683,888, No. 5,741,668, No. 5,777,079, No. 5,804,387, No. 5,874,304, No. 5,876,995, No. 5,925,558). All of the above references are expressly incorporated herein by reference.

Exemplary antigen binding proteins described herein have properties based on a unique epitope on ST2 to which the antigen binding protein binds. The term "epitope" means an amino acid of a target molecule that is in contact with the antigen-binding protein (eg, an antibody) when the antigen-binding protein binds to the target molecule. Epitopes may or may not be contiguous, for example, (i) in a single-chain polypeptide, amino acid residues that are not contiguous with each other in the polypeptide sequence but within the background of the target molecule bind to the antigen-binding protein, or (ii) are included In a multimeric receptor of two or more individual components (e.g., ST2 and AcP), an amino acid residue is present on one or more of the individual components but still binds to the antigen binding protein. An epitope determinant can include a chemically active surface group of a molecule, such as an amino acid, a sugar side chain, a phosphonium group, or a sulfonyl group, and can have specific three dimensional structural characteristics and/or specific charge characteristics. Typically, an antigen binding protein that is specific for a particular target molecule will preferentially recognize an epitope on the target molecule in a complex mixture of proteins and/or macromolecules.

Methods for characterizing epitopes that bind to antigen-binding proteins are well known in the art and include, but are not limited to, binning (cross-competition) (Miller et al., "Epitope binning of murine monoclonal antibodies by a multiplexed pairing assay" J Immunol Methods (2011) 365,118-25), targeting peptides (eg, PEPSPOT ^TM) (Albert et al., "The B-cell Epitope of the Monoclonal Anti-Factor VIII Antibody ESH8 Characterized by peptide Array Analysis " 2008 Thromb Haemost 99,634-7 Mutagenesis methods such as chimera (Song et al., "Epitope Mapping of Ibalizumab, a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity in Infected Patients" J. Virol. (2010) 84, 6935-6942 ), alanine scan (Cunningham and Wells "High-resolution epitope mapping of HGH-receptor interactions by alanine-scanning mutagenesis" Science (1989) 244, 1081-1085), arginine scan (Lim et al., "A diversity of antibody epitopes can induce signaling through the erythropoietin receptor "Biochemistry (2010) 49,3797-3804), HD exchange method (Coates People, "Epitope mapping by amide hydrogen / deuterium exchange coupled with immobilization of antibody, on-line proteolysis, liquid chromatography and mass spectrometry ," Rapid Commun.Mass Spectrom. (2009) 23639-647 ), NMR cross-saturation method (Morgan et al. "Precise epitope mapping of malaria parasite inhibitory antibodies by TROSY NMR cross-saturation" Biochemistry (2005) 44, 518-23) and crystallography (Gerhardt et al., "Structure of IL-17A in complex with a potent, fully human neutralizing antibody" J .Mol. Biol (2009) 394, 905-21). These methods differ in the degree of detail provided by the amino acid containing the epitope.

Antigen binding proteins of the invention include those that overlap with an exemplary antigen binding protein (e.g., Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab30, Ab32, or Ab33) described herein. Episode. In certain embodiments, the antigen binding protein has a consensus epitope with an exemplary antigen binding protein. In other embodiments, the antigen binding protein binds only to the same subgroup of amino acids as the exemplary antigen binding protein.

In certain embodiments, the ST2 antigen binding protein has a consensus or overlapping epitope with Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab30, Ab32 or Ab33 and comprises a) And SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ The amino acid sequence of ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 163, SEQ ID NO: 164 or SEQ ID NO: 165 has at least 90% identity, at least 95 % identical or identical light chain variable domain; b) with SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38. The amino acid sequence set forth in SEQ ID NO: 39, SEQ ID NO: 145, SEQ ID NO: 146 or SEQ ID NO: 147 has at least 90% identity, at least 95% identity or consistent heavy chain. a variable domain; or c) a light chain variable domain of a) and a heavy chain variable domain of b).

In certain embodiments, the ST2 antigen binding protein has a consensus or overlapping epitope with Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab30, Ab32 or Ab33, and comprises The amino acid sequence set forth in SEQ ID NO: 95 has at least 90% identity, at least 95% identity or identity of the light chain variable domain and has at least 90 with the amino acid sequence set forth in SEQ ID NO:29. % identity, at least 95% identity or consensus heavy chain variable domains; these include: at least 90% identity, at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 96 Or a consensus light chain variable domain and a heavy chain variable domain having at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 30; a light chain variable domain having at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 97 and having the amino acid sequence set forth in SEQ ID NO: 31 At least 90% identity, at least 95% identity or consensus heavy chain variable domains; these include: SEQ ID NO: 98 The amino acid sequence having at least 90% identity, at least 95% identity or identity, and at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 32, at least 95% identical or identical heavy chain variable domains; they comprise: a light chain that is at least 90% identical, at least 95% identical or identical to the amino acid sequence set forth in SEQ ID NO: 99 A variable domain and a heavy chain variable domain having at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 33; these include: SEQ ID NO The amino acid sequence of :100 has at least 90% identity, at least 95% identity or one The light chain variable domain and the heavy chain variable domain having at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 34; these include: a light chain variable domain having at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 101 and having at least the amino acid sequence set forth in SEQ ID NO: 35 90% identity, at least 95% identity or consensus heavy chain variable domains; these include: at least 90% identity, at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 102 a spliced or identical light chain variable domain and a heavy chain variable domain having at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 36; a light chain variable domain having at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 103 and to the amino acid sequence set forth in SEQ ID NO: 37 Heavy chain variable domains having at least 90% identity, at least 95% identity, or agreement; these include: SEQ ID NO: 10 The amino acid sequence of 4 having at least 90% identity, at least 95% identity or identity of the light chain variable domain and at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 38, At least 95% identical or identical heavy chain variable domains; these include those that are at least 90% identical, at least 95% identical or consistent to the amino acid sequence set forth in SEQ ID NO: 105 A chain variable domain and a heavy chain variable domain having at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 39; these include: SEQ ID The amino acid sequence of NO: 163 has at least 90% identity, at least 95% identity or identity of the light chain variable domain and is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 145 a heavy chain variable domain that is at least 95% identical or identical; they comprise: at least 90% identity, at least 95% identity or identity to the amino acid sequence set forth in SEQ ID NO: 164 a light chain variable domain and at least 90% identical to at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 146 Or identical to the heavy chain variable domain; and their by comprising the following: and SEQ ID NO: 165 in the amino acid sequence having at least 90% identity, at least 95% identity or A consensus light chain variable domain and a heavy chain variable domain that is at least 90% identical, at least 95% identical or identical to the amino acid sequence set forth in SEQ ID NO:147.

In certain embodiments, the ST2 antigen binding protein has a consensus or overlapping epitope with Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab30, Ab32 or Ab33, and comprises a And SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, The amino acid sequence described in SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 163, SEQ ID NO: 164 or SEQ ID NO: 165 differs by no more than 10 or no more than 5 amino acid added, deleted or substituted light chain variable domains; b) with SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 145, SEQ ID NO: 146 or SEQ ID NO: 147 wherein the amino acid sequence differs by no more than 10 or no more than 5 amino acid addition, deletion or substitution of the heavy chain variable domain; or c) a) the light chain variable domain and b) Heavy chain variable domain.

In certain embodiments, the ST2 antigen binding protein has a consensus or overlapping epitope with Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab30, Ab32 or Ab33, and comprises a light chain variable domain having no more than 10 or no more than 5 amino acid additions, deletions or substitutions in the amino acid sequence set forth in SEQ ID NO: 95 and an amino acid as described in SEQ ID NO:29 A heavy chain variable domain having a sequence that differs by no more than 10 or no more than 5 amino acid additions, deletions or substitutions; these include: a difference of no more than 10 from the amino acid sequence set forth in SEQ ID NO:96 a light chain variable domain with or without more than 5 amino acid additions, deletions or substitutions and no more than 10 or no more than 5 amino acid additions to the amino acid sequence set forth in SEQ ID NO:30, Deletion or substitution of heavy chain variable domains; these include the following: with the amine group described in SEQ ID NO:97 A light chain variable domain having an acid sequence differing by no more than 10 or no more than 5 amino acid additions, deletions or substitutions and no more than 10 or no more than 5 amino acid sequences as described in SEQ ID NO:31 A heavy chain variable domain with added, deleted or substituted amino acids; these include: no more than 10 or no more than 5 amino acid additions to the amino acid sequence set forth in SEQ ID NO:98 , a deleted or substituted light chain variable domain and a heavy chain variable domain that differs from the amino acid sequence set forth in SEQ ID NO: 32 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions And the like: a light chain variable domain that differs from the amino acid sequence set forth in SEQ ID NO: 99 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions and to SEQ ID NO The heavy chain variable domain of the amino acid sequence addition, deletion or substitution differing by no more than 10 or no more than 5 amino acid sequences in 33; these include: as described in SEQ ID NO: 100 a light chain variable domain having an amino acid sequence that differs by no more than 10 or no more than 5 amino acid additions, deletions or substitutions and A heavy chain variable domain that differs from the amino acid sequence set forth in SEQ ID NO: 34 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions; these include: SEQ ID NO: The light chain variable domain in which the amino acid sequence described in 101 differs by no more than 10 or no more than 5 amino acid additions, deletions or substitutions and does not differ from the amino acid sequence described in SEQ ID NO: 35 10 or no more than 5 amino acid added, deleted or substituted heavy chain variable domains; these include: no more than 10 or no more than the amino acid sequence described in SEQ ID NO: 102 5 amino acid added, deleted or substituted light chain variable domains and no more than 10 or no more than 5 amino acid additions, deletions or substitutions to the amino acid sequence set forth in SEQ ID NO:36 Heavy chain variable domains; these include: a light chain variable structure that differs from the amino acid sequence set forth in SEQ ID NO: 103 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions The domain and the amino acid sequence described in SEQ ID NO: 37 differ by no more than 10 or no more than 5 amino acid additions, deletions Substituted heavy chain variable domains; these include: a light chain that differs from the amino acid sequence set forth in SEQ ID NO: 104 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions Variable domain and SEQ ID NO: 38 wherein the amino acid sequence differs by no more than 10 or no more than 5 amino acid additions, deletions or substitutions of heavy chain variable domains; these include: and SEQ ID NO: 105 The amino acid sequence differs by no more than 10 or no more than 5 amino acid added, deleted or substituted light chain variable domains and differs from the amino acid sequence set forth in SEQ ID NO: 39 by no more than 10 Or no more than 5 amino acid added, deleted or substituted heavy chain variable domains; these include: no more than 10 or no more than 5 amino acid sequences as described in SEQ ID NO: 163 An amino acid added, deleted or substituted light chain variable domain and a heavy chain differing from the amino acid sequence set forth in SEQ ID NO: 145 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions Variable domains; these include: a light chain variable domain that differs from the amino acid sequence set forth in SEQ ID NO: 164 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions and No more than 10 or no more than 5 amino acid additions, deletions or subtractions from the amino acid sequence described in SEQ ID NO: 146 Heavy chain variable domains; and these include: a light chain that differs from the amino acid sequence set forth in SEQ ID NO: 165 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions The variable domain and the heavy chain variable domain differing from the amino acid sequence set forth in SEQ ID NO: 147 by no more than 10 or no more than 5 amino acid additions, deletions or substitutions.

In certain embodiments, the ST2 antigen binding protein has a consensus or overlapping epitope with Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab30, Ab32 or Ab33, and comprises The light chain variable domain: a) LCDR1 differing from the LCDR1 sequence described in SEQ ID NO: 106 by no more than 3 amino acid additions, deletions or substitutions; differs from the LCDR2 sequence described in SEQ ID NO: 117 LCDR2 of no more than 3 amino acid additions, deletions or substitutions; and LCDR3 which differs from the LCDR3 sequence described in SEQ ID NO: 128 by no more than 3 amino acid additions, deletions or substitutions; b) and SEQ ID NO: The LCDR1 sequence of 107 does not differ by more than 3 amino acid additions, deletions or substitutions of LCDR1; LCDR2 which differs from the LCDR2 sequence described in SEQ ID NO: 118 by no more than 3 amino acid additions, deletions or substitutions; From the SEQ in the LCDR3 sequence ID NO: 129 LCDR3 having no more than 3 amino acid additions, deletions or substitutions; c) LCDR1 having no more than 3 amino acid additions, deletions or substitutions from the LCDR1 described in SEQ ID NO: 108 An LCDR2 that differs from the LCDR2 sequence described in SEQ ID NO: 119 by no more than three amino acid additions, deletions or substitutions; and no more than three amino acid additions to the LCDR3 sequence described in SEQ ID NO: 130, a missing or substituted LCDR3; d) an LCDR1 that differs from the LCDR1 sequence described in SEQ ID NO: 109 by no more than 3 amino acid additions, deletions or substitutions; and a difference of no more than 3 from the LCDR2 sequence described in SEQ ID NO: 120 LCDR2 with an amino acid added, deleted or substituted; and LCDR3 which differs from the LCDR3 sequence described in SEQ ID NO: 131 by no more than 3 amino acid additions, deletions or substitutions; e) and SEQ ID NO: 110 LCDR1 of LCDR1 sequence differing by no more than 3 amino acid additions, deletions or substitutions; LCDR2 differing from LCDR2 sequence of SEQ ID NO: 121 by no more than 3 amino acid additions, deletions or substitutions; and SEQ ID The LCDR3 sequence described in NO:132 differs by no more than 3 amino acid additions, deletions or substitutions. CDR3; f) LCDR1 differing from the LCDR1 sequence described in SEQ ID NO: 111 by no more than 3 amino acid additions, deletions or substitutions; no more than 3 amino acids differs from the LCDR2 sequence described in SEQ ID NO: 122 Addition, deletion or substitution of LCDR2; and LCDR3 which differs from the LCDR3 sequence described in SEQ ID NO: 133 by no more than 3 amino acid additions, deletions or substitutions; g) differs from the LCDR1 sequence described in SEQ ID NO: 112 LCDR1 with no more than 3 amino acid additions, deletions or substitutions; LCDR2 differing from the LCDR2 sequence described in SEQ ID NO: 123 by no more than 3 amino acid additions, deletions or substitutions; and with SEQ ID NO: 134 The LCDR3 sequence differs by no more than 3 amino acid additions, deletions or substitutions of LCDR3; h) an LCDR1 that differs from the LCDR1 sequence described in SEQ ID NO: 113 by no more than 3 amino acid additions, deletions or substitutions; The LCDR2 sequence described in SEQ ID NO: 124 differs by no more than 3 amino acid additions, deletions or substitutions of LCDR2; and differs from the LCDR3 sequence described in SEQ ID NO: 135 by no more than 3 amino acid additions, deletions or Substituted LCDR3; i) differs from the LCDR1 sequence described in SEQ ID NO: 114 by no more than 3 amino acids Plus, missing Or substituted LCDR1; LCDR2 which differs from the LCDR2 sequence described in SEQ ID NO: 125 by no more than 3 amino acid additions, deletions or substitutions; and no more than 3 amines from the LCDR3 sequence described in SEQ ID NO: 136 LCDR3;j) with an acid addition, deletion or substitution; j) an LCDR1 sequence differing from the LCDR1 sequence described in SEQ ID NO: 115 by no more than 3 amino acid additions, deletions or substitutions; and the LCDR2 sequence described in SEQ ID NO: 126 LCDR2 differing by no more than 3 amino acid additions, deletions or substitutions; and LCDR3; k) and SEQ ID NO differing from the LCDR3 sequence described in SEQ ID NO: 137 by no more than 3 amino acid additions, deletions or substitutions The LCDR1 sequence described in 116: the LCDR1 with no more than 3 amino acid additions, deletions or substitutions; the LCDR2 which differs from the LCDR2 sequence described in SEQ ID NO: 127 by no more than 3 amino acid additions, deletions or substitutions; And LCDR3 which differs from the LCDR3 sequence described in SEQ ID NO: 138 by no more than 3 amino acid additions, deletions or substitutions; l) differs from the LCDR1 sequence described in SEQ ID NO: 166 by no more than 3 amino acid additions , missing or substituted LCDR1; differs from the LCDR2 sequence described in SEQ ID NO: 169 by no more than 3 LCDR2 with amino acid addition, deletion or substitution; and LCDR3 which differs from the LCDR3 sequence described in SEQ ID NO: 172 by no more than 3 amino acid additions, deletions or substitutions; m) and SEQ ID NO: 167 LCDR1 sequence differs by no more than 3 amino acid additions, deletions or substitutions of LCDR1; LCDR2 differs from the LCDR2 sequence described in SEQ ID NO: 170 by no more than 3 amino acid additions, deletions or substitutions; and SEQ ID NO The LCDR3 sequence described in :173 differs by no more than 3 amino acid additions, deletions or substitutions of LCDR3; or n) differs from the LCDR1 sequence described in SEQ ID NO: 168 by no more than 3 amino acid additions, deletions or substitutions LCDR1; LCDR2 differing from the LCDR2 sequence described in SEQ ID NO: 171 by no more than 3 amino acid additions, deletions or substitutions; and no more than 3 amino acids from the LCDR3 sequence described in SEQ ID NO: 174 Addition, deletion or substitution of LCDR3; and heavy chain variable domain comprising: o) HCDR1 differing from the HCDR1 sequence described in SEQ ID NO: 4O by no more than 3 amino acid additions, deletions or substitutions; The HCDR2 sequence described in ID NO: 51 differs by no more than 3 amino acid additions, deletions or deletions. The HCDR2; And HCDR3 differing from the HCDR3 sequence set forth in SEQ ID NO: 62 by no more than 3 amino acid additions, deletions or substitutions; p) differs from the HCDR1 sequence set forth in SEQ ID NO: 41 by no more than 3 amino acid additions , deleted or substituted HCDR1; HCDR2 differing from the HCDR2 sequence set forth in SEQ ID NO: 52 by no more than 3 amino acid additions, deletions or substitutions; and no more than 3 differences from the HCDR3 sequence set forth in SEQ ID NO:63 HCDR3 added, deleted or substituted with an amino acid; q) HCDR1 differing from the HCDR1 sequence described in SEQ ID NO: 42 by no more than 3 amino acid additions, deletions or substitutions; and as described in SEQ ID NO: 53 HCDR2 sequences differ by no more than 3 amino acid additions, deletions or substitutions of HCDR2; and HCDR3 differing from the HCDR3 sequence set forth in SEQ ID NO: 64 by no more than 3 amino acid additions, deletions or substitutions; r) and SEQ The HCDR1 sequence described in ID NO: 43 differs by no more than 3 amino acid additions, deletions or substitutions of HCDR1; and the HCDR2 sequence described in SEQ ID NO: 54 differs by no more than 3 amino acid additions, deletions or substitutions. HCDR2; and the HCDR3 sequence described in SEQ ID NO: 65 differs by no more than 3 amino acid additions, deletions Substituted HCDR3;s) HCDR1 differing from the HCDR1 sequence set forth in SEQ ID NO: 44 by no more than 3 amino acid additions, deletions or substitutions; no more than 3 amines from the HCDR2 sequence set forth in SEQ ID NO: 55 HCDR2 with addition, deletion or substitution of a base acid; and HCDR3 which differs from the HCDR3 sequence described in SEQ ID NO: 66 by no more than 3 amino acid additions, deletions or substitutions; t) and HCDR1 as described in SEQ ID NO: HCDR1 having a sequence difference of no more than 3 amino acid additions, deletions or substitutions; HCDR2 differing from the HCDR2 sequence set forth in SEQ ID NO: 56 by no more than 3 amino acid additions, deletions or substitutions; and SEQ ID NO: The HCDR3 sequence described in 67 differs by no more than 3 amino acid addition, deletion or substitution of HCDR3; u) HCDR1 differs from the HCDR1 sequence described in SEQ ID NO: 46 by no more than 3 amino acid additions, deletions or substitutions ; an HCDR2 that differs from the HCDR2 sequence set forth in SEQ ID NO: 57 by no more than 3 amino acid additions, deletions or substitutions; and no more than 3 amino acid additions to the HCDR3 sequence set forth in SEQ ID NO:68, The deleted or substituted HCDR3; v) differs from the HCDR1 sequence described in SEQ ID NO: 47 by no more than 3 amino acids Addition, deletion or substitution of HCDR1; HCDR2 differing from the HCDR2 sequence set forth in SEQ ID NO: 58 by no more than 3 amino acid additions, deletions or substitutions; and no more than the HCDR3 sequence described in SEQ ID NO: 69 3 amino acid added, deleted or substituted HCDR3; w) HCDR1 differing from the HCDR1 sequence described in SEQ ID NO: 48 by no more than 3 amino acid additions, deletions or substitutions; and SEQ ID NO: 59 The HCDR2 sequence differs by no more than 3 amino acid additions, deletions or substitutions of HCDR2; and HCDR3 differs from the HCDR3 sequence described in SEQ ID NO: 70 by no more than 3 amino acid additions, deletions or substitutions; x) The HCDR1 sequence described in SEQ ID NO: 49 differs by no more than 3 amino acid additions, deletions or substitutions of HCDR1; no more than 3 amino acid additions, deletions or substitutions compared to the HCDR2 sequence set forth in SEQ ID NO:60 HCDR2; and HCDR3 which differs from the HCDR3 sequence set forth in SEQ ID NO: 71 by no more than 3 amino acid additions, deletions or substitutions; y) differs from the HCDR1 sequence described in SEQ ID NO: 50 by no more than 3 amines HCDR1 added, deleted or substituted by a base acid; not comparable to the HCDR2 sequence described in SEQ ID NO: 61 3 amino acid added, deleted or substituted HCDR2; and HCDR3 which differs from the HCDR3 sequence described in SEQ ID NO: 72 by no more than 3 amino acid additions, deletions or substitutions; z) and SEQ ID NO: 148 The HCDR1 sequence differs by no more than 3 amino acid additions, deletions or substitutions of HCDR1; HCDR2 differs from the HCDR2 sequence set forth in SEQ ID NO: 151 by no more than 3 amino acid additions, deletions or substitutions; The HCDR3 sequence described in ID NO: 154 differs by no more than 3 amino acid additions, deletions or substitutions of HCDR3; aa) differs from the HCDR1 sequence described in SEQ ID NO: 149 by no more than 3 amino acid additions, deletions or Substituted HCDR1; HCDR2 differing from the HCDR2 sequence set forth in SEQ ID NO: 152 by no more than 3 amino acid additions, deletions or substitutions; and no more than 3 amine groups from the HCDR3 sequence set forth in SEQ ID NO: 155 Acid-added, deleted or substituted HCDR3; or bb) HCDR1 differing from the HCDR1 sequence described in SEQ ID NO: 150 by no more than 3 amino acid additions, deletions or substitutions; and HCDR2 sequences as described in SEQ ID NO: 153 HCDR2 with no more than 3 amino acid additions, deletions or substitutions; and SEQ ID The HCDR3 sequences described in NO: 156 differ by no more than 3 amino acid additions, deletions or substitutions of HCDR3.

Preferred ST2 antigen binding proteins set forth above include those comprising the light chain variable domain of a) and the heavy chain variable domain of o); they comprise the light chain variable domain of b) and a heavy chain variable domain; those comprising the light chain variable domain of c) and the heavy chain variable domain of q); they comprise the light chain variable domain of d) and r) Heavy chain variable domains; those comprising the light chain variable domain of e) and the heavy chain variable domain of s); they comprise the light chain variable domain of f) and the heavy chain of t) Variable domain; those comprising the light chain variable domain of g) and the heavy chain variable domain of u); they comprise the light chain variable domain of h) and the heavy chain of v) Domains; those comprising the light chain variable domain of i) and the heavy chain variable domain of w); they comprise the light chain variable domain of j) and the heavy chain variable domain of x) Those comprising the light chain variable domain of k) and the heavy chain variable domain of y); those comprising the light chain variable domain of l) and the heavy chain variable domain of z); These include the light chain variable domain of m) and the heavy chain variable structure of aa) Persons; and their light chain variable domain comprising n), and bb) the heavy chain variable domain persons.

Antigen-binding proteins with a consensus epitope or overlapping epitopes typically bind to the antigen by cross-competition. Thus, in certain embodiments, an antigen binding protein of the invention competes with Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab30, Ab32 or Ab33. "Cross-compete or cross-competition" means that the antigen-binding protein competes for the same epitope or binding site on the target. This competition can be determined by the following analysis: Reference antigen binding proteins (eg, antibodies or antigen binding portions) prevent or inhibit the specific binding of the test antigen binding protein and vice versa. A variety of competitive binding assays can be used to determine whether a test molecule competes for binding to a reference molecule. Examples of assays that may be employed include solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich-type competition analysis (see, for example, Stahli et al. (1983) Methods in Enzymology 9: 242-253), solid phase direct biotin-avidin EIA (see, for example, Kirkland et al, (1986) J. Immunol. 137:3614-3619), solid phase direct labeling analysis, solid phase direct labeling sandwich Analysis, Luminex (Jia et al., "A novel method of Multiplexed Competitive Antibody Binning for the characterization of monoclonal antibodies" J. Immunological Methods (2004) 288, 91-98) and surface plasma resonance ((Song et al., "Epitope Mapping of Ibalizumab, a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity in Infected Patients" J. Virol. (2010) 84, 6935-6942). An exemplary method for determining cross-competition is set forth in Example 5. Usually When the competitive antigen binding protein is present in excess, it inhibits binding of the reference antigen binding protein to a common antigen by at least 50%, 55%, 60%, 65%, 70% or 75%. In some cases The inhibition of binding at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more.

Ab2 binds human and cynomolgus ST2 with high affinity and blocks IL-33 binding to ST2, thereby blocking IL-33 mediated ST2 signaling. Antibodies that have identical, similar or overlapping epitopes to Ab2 share these unique characteristics. In a preferred embodiment, the ST2 antigen binding protein competes with Ab2 for binding to ST2. Exemplary ST2 antigen binding proteins that compete with Ab2 include Ab1, Ab3, Ab5, Ab7, Ab8, and Ab30 (see Example 5). If an attempt is made to find an antibody that binds to an epitope that is overlapping, similar or identical to Ab2, one or more antibodies can be screened for cross-over competition with Ab2. Furthermore, when a variant of an antibody that cross-reacts with Ab2 is made, these antibodies can be screened to determine whether cross-competition is maintained after the mutation, indicating that the epitope of the variant is not significantly altered from the parent molecule. Thus, in certain embodiments, the invention provides for antibody variants that compete with Ab2 for binding to ST2.

In addition to competing with each other, antibodies with overlapping, similar or identical epitopes can also be affected by ST2 mutagenesis in a similar manner. Certain mutations can inhibit the binding of antibodies; others can enhance or activate binding. In Example 11, a portion of the extracellular domain of ST2 was scanned for arginine/alanine mutagenesis and assayed for effects on exemplary antibodies. Included within the scope of the invention The ST2 binding protein has the following properties such that it is subjected to mutagenesis in a manner similar to the exemplary antibody.

In certain embodiments, the binding of the ST2 antigen binding protein is inhibited by a single mutation in ST2, wherein the single mutation is selected from the group consisting of L14R, I15R, S33R, E43R, V47R, A62R, G65R, T79R, D92R, D97R, V104R, G138R, N152R and V176R. In a preferred embodiment, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more in the group A single mutation of one, eight or more, nine or more, ten or more, or all individually inhibits binding of the ST2 binding protein. In other embodiments, the binding of the ST2 antigen binding protein is activated by a single mutation in ST2, wherein the single mutation is selected from the group consisting of L53R, R72A, and S73R. In a preferred embodiment, all of the single mutations in the population individually activate the binding of the ST2 binding protein. In a preferred embodiment, the ST2 antigen binding protein shares the property of Ab2 and is inhibited by any of L14R, I15R, S33R, E43R, V47R, A62R, G65R, T79R, D92R, D97R, V104R, G138R, N152R and V176R. And activated by any of L53R, R72A, and S73R.

Another method for characterizing antibodies based on epitopes is indole hydrogen/deuterium exchange (HDX). HDX has been widely used to study protein conformation and kinetics, protein-ligand interactions, and protein-protein interactions (Zhang and Smith 1993, Engen and Smith 2001). Mass spectrometry provides a powerful tool for determining the degree of exchange, since replacing the single hydrogen with hydrazine increases the mass of each exchange by 1 Da. The extent of HDX can be readily measured at the peptide level by analysis of proteolytic digests by liquid chromatography coupled with tandem mass spectrometry under controlled conditions (Engen and Smith 2001, Baerga-Ortiz, Hughes et al. 2002, Codreanu, Ladner et al. 2002, Hamuro, Coales et al. 2006, Coales, Tuske et al. 2009, Zhang, Zhang et al. 2012).

Comparing ST2 protein water in the absence and presence of antibodies (free pair binding) Resolving the antigen HDX profile between digests reveals interaction sites. Specifically, when the antibody binds to ST2, the solvent in the free ST2 and the indoleamine hydrogen can be protected, and therefore, a slower exchange rate is observed. Thus, a potential binding epitope in the presence of an antibody that is less abundant than in its absence is obtained. When determining epitopes, other factors are considered, including exchange rate in the free state, knowledge of the structure of the antigenic protein, and results from other epitope mapping efforts.

The binding of Ab2 to T2 was analyzed by HDX as described in Example 12. Analysis confirmed that Ab2 binds to the amino acid 33-44 and 88-94 of the ST2 structure comprising the amino acid 19-322 of SEQ ID NO: 1 (amino acids 15-26 and 70-76, respectively, of mature ST2). ) / Change its exchange rate. Antibodies having overlapping epitopes, similar or identical epitopes to Ab2 will also bind to the amino acid in SEQ ID NO: 1 and 33-44 and 88-94 to alter the rate of exchange. In certain embodiments, the ST2 binding protein (eg, an antibody) protects any of the amino acids 33-44 of SEQ ID NO: 1 when bound to ST2 and analyzed by HDX. In other embodiments, any of the amino acids 88-94 is protected. Both indicate that the binding epitope overlaps with Ab2. In a preferred embodiment, either of 33-44 is protected from either of 88-94. In certain embodiments, the ST2 binding protein (eg, an antibody) protects all of the amino acids 33-44 of SEQ ID NO: 1 when bound to ST2 and analyzed by HDX. In other embodiments, the entire amino acid 88-94 is protected. Both indicate that they have a similar binding epitope to Ab2. In the preferred embodiment, all 33-44 and all 88-94 are protected, indicating an epitope that is identical or nearly identical to Ab2.

The binding of Ab2 to ST2 was further analyzed using X-ray crystallography. X-ray crystallography is consistent with HDX analysis. The interface between Ab and antigen can be determined/defined in a number of ways. In Example 13, the interface was measured using solvent exposure and determined by distance. In, for example, by solvent exposure difference or less than 5 The distance is determined by the ST2 residue in the interface with Ab2 (corresponding to the position in mature ST2 (lack of leader sequence)) K1, F2, P19, R20, Q21, G22, K23, Y26, I70, V71, R72 , S73, P74, T75, F76, N77, R78, T79 and Y81. In certain embodiments, the ST2 binding protein forms an interface with ST2 that overlaps with the interface of Ab2, including those of K1, F2, P19, R20, Q21, G22, K23, Y26, I70, V71, R72, S73, P74 Any one of T75, F76, N77, R78, T79 or Y81 is within the interface. In some embodiments, the ST2 binding protein forms an interface with ST2, wherein P19, R20, Q21, G22, K23, and/or Y26 are within the interface. In other embodiments, I70, V71, R72, S73, P74, T75, F76, N77, R78, T79, and/or Y81 are within the interface. In the preferred embodiment, K1, F2, P19, R20, Q21, G22, K23, Y26, I70, V71, R72, S73, P74, T75, F76, N77, R78, T79 and Y81 are within the interface.

The crystal structure indicates that certain amino acid residues form hydrogen bonds or salt bridges with the amino acid of Ab2. Their residues include K1, R20, K23, Y26, T75, N77, R78 and T79. In certain embodiments, the ST2 antigen binding protein forms a hydrogen bond or a salt bridge with one or more of K1, R20, K23, Y26, T75, N77, R78, and T79.

The crystal structure further provides information on which residues of Ab2 form an interface with ST2. Figure 10 indicates residues in the light chain variable region and the heavy chain variable region that form an interface with ST2. Also indicated is a residue that forms a hydrogen bond or a salt bridge with the amino acid in ST2. This information can be used to design variants of Ab2, including those that have 90% identity, 95% identity, and 10 or fewer of the light or heavy chain variable domains of Ab2. Variable domains that are inserted, deleted, and/or substituted. It may be desirable to maintain the amino acid in the interface while changing the non-interface residues. Thus, an Ab2 variant having one or more amino acid additions, substitutions and/or deletions in one or more of the CDRs of Ab2 that maintains binding to ST2 can be designed and produced.

In some embodiments, the ST2 binding protein comprises a variant of the Ab2 light chain variable region (SEQ ID NO: 96), wherein D28, I29, S30, N31, Y32, Y49, D50, N53, E55, T56, D91, D92, N93, F94 and/or L96 remain unchanged or comprise conservative substitutions thereof; and/or variants of the Ab2 heavy chain variable region (SEQ ID NO: 30), wherein W33, I50, D57, R59, H99, G100 , T101, S102, S103, D104, Y105 and/or Y106 remains unchanged or contains conservative mutations. In the preferred embodiment, D28, N31, D50, N53, E55, D91, and D92 of the light chain variable region remain unchanged and S102, S103, D104, and Y105 of the heavy chain remain unchanged.

Polynucleotide encoding ST2 antigen binding protein

Nucleic acids encoding ST2 antigen binding proteins, including antibodies, are encompassed within the invention, as defined herein. Preferred nucleic acids include those encoding the exemplary light and heavy chains described herein.

An exemplary nucleic acid encoding Ab1 LC comprises a nucleic acid of the sequence set forth in SEQ ID NO:73.

An exemplary nucleic acid encoding Ab2 LC comprises a nucleic acid of the sequence set forth in SEQ ID NO:74.

An exemplary nucleic acid encoding Ab3 LC comprises a nucleic acid of the sequence set forth in SEQ ID NO:75.

An exemplary nucleic acid encoding Ab4 LC comprises a nucleic acid of the sequence set forth in SEQ ID NO:76.

An exemplary nucleic acid encoding Ab5 LC comprises a nucleic acid of the sequence set forth in SEQ ID NO:77.

An exemplary nucleic acid encoding Ab6 LC comprises a nucleic acid of the sequence set forth in SEQ ID NO:78.

An exemplary nucleic acid encoding Ab7 LC comprises a nucleic acid of the sequence set forth in SEQ ID NO:79.

An exemplary nucleic acid encoding Ab8 LC comprises a nucleic acid of the sequence set forth in SEQ ID NO:80.

An exemplary nucleic acid encoding Ab9 LC comprises a nucleic acid of the sequence set forth in SEQ ID NO:81.

An exemplary nucleic acid encoding Ab10 LC comprises a nucleic acid of the sequence set forth in SEQ ID NO:82.

An exemplary nucleic acid encoding Ab11 LC comprises a nucleic acid of the sequence set forth in SEQ ID NO:83.

An exemplary nucleic acid encoding Ab30 LC comprises a nucleic acid of the sequence set forth in SEQ ID NO:157.

An exemplary nucleic acid encoding Ab32 LC comprises a nucleic acid of the sequence set forth in SEQ ID NO:158.

An exemplary nucleic acid encoding Ab33 LC comprises a nucleic acid of the sequence set forth in SEQ ID NO:159.

An exemplary nucleic acid encoding Ab1 HC comprises a nucleic acid of the sequence set forth in SEQ ID NO: 7.

An exemplary nucleic acid encoding Ab2 HC comprises a nucleic acid of the sequence set forth in SEQ ID NO:8.

An exemplary nucleic acid encoding Ab3 HC comprises a nucleic acid of the sequence set forth in SEQ ID NO:9.

An exemplary nucleic acid encoding Ab4 HC comprises a nucleic acid of the sequence set forth in SEQ ID NO: 10.

An exemplary nucleic acid encoding Ab5 HC comprises a nucleic acid of the sequence set forth in SEQ ID NO:11.

An exemplary nucleic acid encoding Ab6 HC comprises a nucleic acid of the sequence set forth in SEQ ID NO: 12.

An exemplary nucleic acid encoding Ab7 HC comprises a nucleic acid of the sequence set forth in SEQ ID NO: 13.

An exemplary nucleic acid encoding Ab8 HC comprises a nucleic acid of the sequence set forth in SEQ ID NO: 14.

An exemplary nucleic acid encoding Ab9 HC comprises a nucleic acid of the sequence set forth in SEQ ID NO: 15.

An exemplary nucleic acid encoding Ab10 HC comprises a nucleic acid of the sequence set forth in SEQ ID NO: 16.

An exemplary nucleic acid encoding Ab11 HC comprises a nucleic acid of the sequence set forth in SEQ ID NO:17.

An exemplary nucleic acid encoding Ab30 HC comprises a nucleic acid of the sequence set forth in SEQ ID NO:139.

An exemplary nucleic acid encoding Ab32 HC comprises a nucleic acid of the sequence set forth in SEQ ID NO:140.

An exemplary nucleic acid encoding Ab33 HC comprises a nucleic acid of the sequence set forth in SEQ ID NO:141.

Aspects of the invention include polynucleotide variants encoding the amino acid sequences described herein (e.g., due to degeneracy).

The present invention includes various embodiments including, but not limited to, the following example embodiments.

An isolated polynucleotide, wherein the polynucleotide encodes one or more polypeptides comprising an amino acid sequence selected from the group consisting of: A. 1. A light chain variable domain sequence that is at least 90% identical to the light chain variable domain sequence set forth in SEQ ID NOs: 95-105, 163-165; 2. A heavy chain variable domain sequence that is at least 90% identical to the heavy chain variable domain sequence set forth in SEQ ID NOs: 29-39, 145-147; 3. (1) a light chain variable domain and (2) a heavy chain variable domain; B. a light chain variable domain comprising CDR1, CDR2, CDR3 and/or a heavy chain variable domain comprising CDR1, CDR2, CDR3, wherein each CDR of CDR1, CDR2, CDR3 is identical or different from the following sequence A total of 3 amino acid additions, substitutions and/or deletions: 1. Ab1 light chain CDR1 (SEQ ID NO: 106), CDR2 (SEQ ID NO: 117), CDR3 (SEQ ID NO: 128) or heavy chain CDR1 (SEQ ID NO: 40), CDR2 (SEQ ID NO: 51), CDR3 (SEQ ID NO: 62); 2. Light chain CDR1 of Ab2 (SEQ ID NO: 107), CDR2 (SEQ ID NO: 118), CDR3 (SEQ ID NO: 129) or heavy chain CDR1 (SEQ ID NO: 41), CDR2 (SEQ ID NO: 52), CDR3 (SEQ ID NO: 63); 3. Light chain CDR1 (SEQ ID NO: 108), CDR2 (SEQ ID NO: 119), CDR3 (SEQ ID NO: 130) or heavy chain CDR1 (SEQ ID NO: 42), CDR2 of Ab3 (SEQ ID NO: 53), CDR3 (SEQ ID NO: 64); 4. Light chain CDR1 (SEQ ID NO: 109), CDR2 (SEQ ID NO: 120), CDR3 (SEQ ID NO: 131) of Ab4 or Heavy chain CDR1 (SEQ ID NO: 43), CDR2 (SEQ ID NO: 54), CDR3 (SEQ ID NO: 65); 5. Light chain CDR1 (SEQ ID NO: 110), CDR2 (SEQ ID NO: 121), CDR3 (SEQ ID NO: 132) or heavy chain CDR1 (SEQ ID NO: 44), CDR2 (SEQ ID NO: 55), CDR3 (SEQ ID NO: 66); 6. Light chain CDR1 of Ab6 (SEQ ID NO: 111), CDR2 (SEQ ID NO: 122), CDR3 (SEQ ID NO: 133) or heavy chain CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 56), CDR3 (SEQ ID NO: 67); 7. Light chain CDR1 (SEQ ID NO: 112), CDR2 (SEQ ID NO: 123), CDR3 (SEQ ID NO) of Ab7 134) or heavy chain CDR1 (SEQ ID NO: 46), CDR2 (SEQ ID NO: 57), CDR3 (SEQ ID NO: 68); 8. Ab8 light chain CDR1 (SEQ ID NO: 113), CDR2 (SEQ ID NO: 124), CDR3 (SEQ ID NO: 135) or heavy chain CDR1 (SEQ ID NO: 47), CDR2 (SEQ ID NO: 58), CDR3 (SEQ ID NO: 69); 9. Light chain of Ab9 CDR1 (SEQ ID NO: 114), CDR2 (SEQ ID NO: 125), CDR3 (SEQ ID NO: 136) or heavy chain CDR1 (SEQ ID NO: 48), CDR2 (SEQ ID NO: 59), CDR3 (SEQ ID NO: 70); 10. Light chain CDR1 (SEQ ID NO: 115), CDR2 (SEQ ID NO: 126), CDR3 (SEQ ID NO: 137) or heavy chain CDR1 (SEQ ID NO: 49), CDR2 (SEQ ID NO: 60), CDR3 (SEQ ID NO: 71); 11. Light chain CDR1 (SEQ ID NO: 116), CDR2 (SEQ ID NO: 127), CDR3 (SEQ ID NO: 138) or heavy chain CDR1 (SEQ) of Ab11 ID NO: 50), CDR2 (SEQ ID NO: 61), CDR3 (SEQ ID NO: 72); 12. Light chain CDR1 (SEQ ID NO: 166), CDR2 (SEQ ID NO: 169), CDR3 of Ab30 SEQ ID NO: 172) or heavy chain CDR1 (SEQ ID NO: 148), CDR2 (SEQ ID NO: 151), CDR3 (SEQ ID NO: 154); 13. Light chain CDR1 of Ab32 (SEQ ID NO: 167) , CDR2 (SEQ ID NO: 170), CDR3 (SEQ ID NO: 173) or heavy chain CDR1 (SEQ ID NO: 149), CDR2 (SEQ ID NO: 152), CDR3 (SEQ ID NO: 155); . Light chain CDR1 (SEQ ID NO: 168), CDR2 (SEQ ID NO: 171), CDR3 (SEQ ID NO: 174) or heavy chain CDR1 (SEQ ID NO: 150), CDR2 (SEQ ID NO: 153) ), CDR3 (SEQ ID NO: 156).

In a preferred embodiment, the polypeptide encoded by the isolated nucleic acid binds to a component of the antigen binding protein of ST2.

A nucleotide sequence corresponding to the amino acid sequence described herein (for use as a probe or primer for isolating nucleic acids or as a query sequence for database searches) can be "returned" from an amino acid sequence or by discrimination Obtained from the region where the polypeptide encoding the DNA sequence has an amino acid identity. A known polymerase chain reaction (PCR) program can be used to isolate and amplify a DNA sequence encoding a desired combination of ST2 antigen binding protein or ST2 antigen binding protein polypeptide fragments. Oligonucleotides that define the desired ends of the DNA fragment combination are used as 5' and 3' primers. The oligonucleotide may additionally contain a restriction endonuclease recognition site to facilitate insertion of the amplified combination of DNA fragments into the expression vector. PCR techniques are described in Saiki et al, Science 239:487 (1988); Recombinant DNA Methodology ; Wu et al., eds., Academic Press, San Diego (1989), pp. 189-196; and PCR Protocols : A Guide to Methods and Applications , edited by Innis et al., Academic Press (1990).

The nucleic acid molecules of the invention include both DNA and RNA in both single-stranded and double-stranded forms, as well as corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. The nucleic acid molecules of the invention include full length gene or cDNA molecules and combinations of fragments thereof. The nucleic acids of the invention are preferably derived from human sources, but the invention also includes those derived from non-human species.

In the case of a nucleic acid isolated from a naturally occurring source, an "isolated nucleic acid" is a nucleic acid that has been separated from adjacent genetic sequences in the genome of the organism from which the nucleic acid is isolated. In the case of, for example, a nucleic acid (eg, a PCR product, a cDNA molecule, or an oligonucleotide) that is enzymatically synthesized or chemically synthesized from a template, it is understood that the nucleic acid obtained from such processes is subjected to isolation of the nucleic acid. An isolated nucleic acid molecule refers to a nucleic acid molecule that is in the form of a separate fragment or as a component of a larger nucleic acid construct. In a preferred embodiment, the nucleic acid is substantially free of contaminating endogenous materials. Nucleic acid molecules have preferably been prepared by standard biochemical methods (e.g., as summarized in Sambrook et al, Molecular Cloning: A Laboratory Manual , 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). At least one isolated DNA or RNA is obtained in a substantially pure form and in an amount or concentration that enables the identification, manipulation and recovery of its constitutive nucleotide sequence. Preferably, such sequences are provided and/or constructed in the form of open reading frames that are not interrupted by internal non-translated sequences or introns that are normally found in eukaryotic genes. The sequence of non-translated DNA can be stored 5' or 3' in the open reading frame, wherein the sequences do not interfere with manipulation or expression of the coding region.

The invention also encompasses nucleic acids that hybridize under moderately stringent conditions, and more preferably under highly stringent conditions, to a nucleic acid encoding a ST2 antigen binding protein as described herein. The basic parameters affecting the selection of hybridization conditions and guidelines for designing suitable conditions are set forth in Sambrook, Fritsch and Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, vol. 9). Chapter and Chapter 11; and Current Protocols in Molecular Biology, 1995, edited by Ausubel et al., John Wiley & Sons, Inc., Sections 2.10 and 6.3-6.4), and may be based on, for example, the length of DNA and / or base composition is easily determined. One way to achieve moderately stringent conditions involves the use of a pre-wash solution containing 5 x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), about 50% methotrexate, 6 x SSC hybridization buffer, and about 55 ° C. Hybridization temperature (or other similar hybridization solution, such as those containing about 50% methotrexate, and the hybridization temperature is about 42 ° C) and about 60 ° C in 0.5 x SSC, 0.1% SDS. Generally, highly stringent conditions are defined as the above hybridization conditions, but washed at about 68 ° C, 0.2 x SSC, 0.1% SDS. SSPE (1×SSPE 0.15M NaCl, 10 mM NaH ₂ PO ₄ and 1.25 mM EDTA, pH 7.4) can replace SSC in the hybridization and washing buffer (1×SSC system 0.15 M NaCl and 15 mM sodium citrate); Washing was carried out 15 minutes after hybridization. It will be appreciated that the wash temperature and wash salt concentration can be adjusted as needed to achieve the desired stringency by applying the basic principles of controlled hybridization reactions and duplex stability, as is known to those skilled in the art and as further described below. (See, for example, Sambrook et al., 1989). When a nucleic acid is hybridized to a target nucleic acid of an unknown sequence, the length of the hybrid is assumed to be the length of the hybrid nucleic acid. When a nucleic acid of known sequence is hybridized, the length of the hybrid can be determined by comparing the sequences of the nucleic acids and identifying one or more regions of optimal sequence complementarity. Hybridization temperatures of hybrids of less than 50 base pairs in length are expected to be 5 to 10 ° C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equation. For hybrids less than 18 base pairs in length, Tm (°C) = 2 (number of A + T bases) + 4 (number of G + C bases). For hybrids greater than 18 base pairs in length, Tm(°C)=81.5+16.6(log ₁₀ [Na ⁺ ])+0.41(G+C%)-(600/N), where N-type hybrids The number of bases in the middle, and the concentration of sodium ions in the [Na ⁺ ] hybridization buffer ([X ⁺ ] = 0.165 M of 1 × SSC). Preferably, each such hybrid nucleic acid is at least 15 nucleotides in length (or better than at least 18 nucleotides or at least 20 nucleotides or at least 25 nucleotides or at least 30 nucleotides or At least 40 nucleotides or optimally at least 50 nucleotides), or at least 25% (more preferably at least 50% or at least 60% or at least 70% and most preferably at least 50% of the length of the nucleic acid of the invention to which it is hybridized) 80%), and the nucleic acid of the invention to which it is hybridized has at least 60% (more preferably at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85) %, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, At least 98% or at least 99% and optimally at least 99.5%) sequence identity, wherein sequence identity is determined by comparing sequences of hybridized nucleic acids when aligning to maximize overlap and identity while minimizing sequence vacancies , as explained in more detail above.

Variants of the invention are typically prepared by performing site-specific mutagenesis of the nucleotides of the DNA encoding the antigen-binding protein using a cassette or PCR mutagenesis or other techniques well known in the art to generate DNA-encoding variants. , and thereafter, the recombinant DNA is expressed in cell culture as outlined herein. However, antigen-binding protein fragments comprising variant CDRs having up to about 100-150 residues can be prepared by in vitro synthesis using defined techniques. Variants typically exhibit the same qualitative biological activity as a naturally occurring analog, for example, in combination with ST2, but may also have variants with modified properties, as outlined more fully below.

As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, a very large number of nucleic acids can be prepared, all of which encode the CDRs of the invention (as well as other components of the heavy and light chain or antigen binding proteins). . Thus, specific amino acid sequences have been identified, and those skilled in the art can achieve any number of differences by simply modifying the sequence of one or more codons without altering the amino acid sequence of the encoded protein. Nucleic acid.

The invention also provides expression systems and constructs in the form of plasmids, expression vectors, transcriptional or expression cassettes comprising at least one additional nucleotide as described above. Additionally, the invention provides host cells comprising such expression systems or constructs.

Generally, the expression vector used in any host cell can be used for plasmid maintenance and A sequence for the selection and expression of an exogenous nucleotide sequence. Such sequences (collectively referred to as "flank sequences") will generally include, in certain embodiments, one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a sequence of transcription termination sequences a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, and a nucleic acid for inserting a polypeptide encoding the polypeptide to be expressed Connector region and optional marker elements. Each of these sequences is discussed below.

The vector may optionally contain a "tag" coding sequence, ie, an oligonucleotide molecule positioned at the 5' or 3' end of the ST2 antigen binding protein coding sequence; the oligonucleotide sequence encoding a poly-His (eg, six His) or Another "tag", such as FLAG, HA (hemagglutinin influenza virus) or myc , therefore, there are commercially available antibodies. This tag is typically fused to the polypeptide when expressed by the polypeptide and can be used as a means of affinity purification or detection of the ST2 antigen binding protein from the host cell. For example, affinity purification can be accomplished by column chromatography using an antibody against the tag as an affinity matrix. Optionally, the tag can then be removed from the purified ST2 antigen binding protein by various means, such as the use of certain cleavage peptidases.

The flanking sequences can be homologous (ie, from the same species and/or strain as the host cell), heterologous (ie, from a species different from the host cell species or strain), hybrid (ie, from more than one source) Combination of flanking sequences), synthetic or natural. Thus, the source of the flanking sequence can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism or any plant, provided that the flanking sequence functions in the host cell machinery and can be hosted by the host Cell machine activation.

The flanking sequences useful in the vectors of the present invention can be obtained by any of a number of methods well known in the art. Generally, the flanking sequences useful herein have previously been identified by localization and/or by restriction endonuclease digestion and thus can be isolated from appropriate tissue sources using suitable restriction endonucleases. In some cases, the entire nucleotide sequence of the flanking sequence is known. Herein, the flanking sequences can be synthesized using the methods described herein for nucleic acid synthesis or colonization.

Whether all or only a portion of the flanking sequences are known, they can utilize polymerase chain reaction (PCR) and/or by using appropriate probes (eg, oligonucleotides and/or flanks from the same or another species) Sequence fragments) were screened for genomic libraries. If the flanking sequence is not known, the DNA fragment containing the flanking sequence can be isolated from a larger piece of DNA that can contain, for example, a coding sequence or even other genes. Isolation can be achieved by restriction endonuclease digestion to generate the appropriate DNA fragment, followed by agarose gel purification, Qiagen® column chromatography (Chatsworth, CA) or other methods known to those skilled in the art. Separate. Those skilled in the art should readily appreciate the selection of suitable enzymes for this purpose.

The origin of replication is typically a portion of the prokaryotic expression vector that they have purchased, and this origin facilitates amplification of the vector in the host cell. If the vector of choice does not contain an origin of replication, it can be chemically synthesized and ligated into the vector according to known sequences. For example, the origin of replication of plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most Gram-negative bacteria, and each viral origin (eg, SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV)) Or papillomaviruses, such as HPV or BPV, can be used to select vectors in mammalian cells. In general, mammalian expression vectors do not require an origin of replication component (e.g., the SV40 origin is typically used, simply because it also contains an early promoter).

The transcription termination sequence is typically located 3' to the end of the polypeptide coding region and can be used to terminate transcription. Typically, the transcription termination sequences in prokaryotic cells are sequentially G-C rich fragments as well as polyT sequences. Although the sequence may be readily selected from the library or even partially purchased as a vector, it may also be synthesized using methods for nucleic acid synthesis (e.g., as described herein).

The selectable marker gene encodes a protein required for the survival and growth of host cells grown in a selective medium. A typical selection marker gene encodes a protein that: (a) confers resistance to a prokaryotic host cell to an antibiotic or other toxin (eg, ampicillin, tetracycline, and vanamycin); (b) supplements the nutrients of the cell Insufficient defect; or (c) a key nutrient that is not available from the complex medium or defined medium. specific The selectable markers are the oxymycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. Advantageously, the neomycin resistance gene can also be used in selection in prokaryotic and eukaryotic host cells.

Other alternative genes can be used to amplify the genes that will be expressed. Amplification lines produce a process in which the genes required for growth or cell survival are tandemly repeated in the chromosomes of successive generations of recombinant cells. Examples of selectable markers suitable for mammalian cells include dihydrofolate reductase (DHFR) and promoterless thymidine kinase. Mammalian cell transformants are placed under selective pressure, wherein only these transformants are uniquely suitable for survival by means of an alternative gene present in the vector. The selection pressure is applied by culturing the transformed cells under conditions in which the concentration of the selection reagent in the medium is continuously increased, thereby amplifying the alternative gene and encoding another gene (for example, an antigen-binding protein antibody that binds to the ST2 polypeptide). DNA. Thus, an increased amount of a polypeptide such as an ST2 antigen binding protein is synthesized from the amplified DNA.

The ribosome binding site is typically required for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryote) or a Kozak sequence (eukaryote). This element is typically located at the 3' end of the promoter and is to be subjected to the 5' end of the coding sequence of the expressed polypeptide. In certain embodiments, one or more coding regions can be operably linked to an internal ribosome binding site (IRES), allowing translation of two open reading frames from a single RNA transcript.

In some cases, such as if glycosylation is desired in a eukaryotic host cell expression system, various pre- or pro-sequences can be manipulated to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide can be altered or the original sequence added, which can also affect glycosylation. The final protein product may have one or more other amino acids that may not be completely removed by performance in the -1 position (relative to the first amino acid of the mature protein). For example, the final protein product can have one or two amino acid residues attached to the amine terminus found in the peptidase cleavage site. Alternatively, if the enzyme cleaves at this region within the mature polypeptide, the use of some enzymatic cleavage sites can result in a slightly truncated form of the desired polypeptide.

The expression and selection vector of the present invention usually contain a recognition by the host organism and The mode of operation is linked to a promoter encoding a molecule of the ST2 antigen binding protein. The promoter is located upstream of the initiation codon (i.e., 5') of the structural gene (typically within about 100 to 1000 bp) to control the transcription of the structural gene. Traditionally, promoters have been classified into one of two categories: inducible promoters and constitutive promoters. An inducible promoter can initiate an increase in the amount of transcription of DNA under its control in response to a change in culture conditions (eg, presence or absence of nutrients or temperature changes). On the other hand, a constitutive promoter evenly transcribes a gene operably linked thereto, i.e., has little or no control over gene expression. A large number of promoters recognized by a variety of potential host cells are well known in the art. A suitable promoter is operably linked to a DNA encoding a ST2 antigen-binding protein of the invention comprising a heavy or light chain by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector by: .

Suitable promoters for use with yeast hosts are also well known in the art. Advantageously, the yeast enhancer is used with a yeast promoter. Suitable promoters for use with mammalian host cells are well known in the art and include, but are not limited to, those derived from viruses such as polyomavirus, fowlpox virus, adenovirus (eg, adenovirus 2) ), bovine papilloma virus, avian sarcoma virus, giant cell virus, retrovirus, hepatitis B virus and most preferably prion 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, for example, heat shock promoters and actin promoters.

Other promoters of interest include, but are not limited to, the SV40 early promoter (Benoist and Chambon, 1981, Nature 290: 304-310); the CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. USA 81) :659-663); a promoter contained within the 3' long-end repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22: 787-797); herpes thymidine kinase promoter (Wagner Et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1444-1445); promoters and regulatory sequences derived from the metallothionein gene (Prinster et al., 1982, Nature 296: 39-42); A prokaryotic promoter such as an endoprolyl promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731); or a tac promoter (DeBoer et al., 1983, Proc. Natl. Acad.Sci.USA 80:21-25). Also contemplated are the following animal transcriptional control regions that exhibit tissue specificity and have been used in transgenic animals: an elastase I gene control region active in pancreatic acinar cells (Swift et al., 1984, Cell 38: 639-646; Ornitz et al. Human, 1986, Cold Spring Harbor Symp. Quant. Biol . 50: 399-409; MacDonald, 1987, Hepatology 7: 425-515); Insulin Gene Control Region Active in Pancreatic β Cells (Hanahan, 1985, Nature 315: 115-122); an immunoglobulin gene control region active in lymphoid cells (Grosschedl et al, 1984, Cell 38: 647-658; Adames et al, 1985, Nature 318: 533-538; Alexander et al. Human, 1987, Mol. Cell. Biol. 7: 1436-1444); mouse mammary tumor virus control region active in testicular cells, breast cells, lymphoid cells and mast cells (Leder et al., 1986, Cell 45) : 485-495); an active albumin gene control region in the liver (Pinkert et al., 1987, Genes and Devel. 1: 268-276); an active alpha-fetoprotein gene control region in the liver (Krumlauf et al., 1985, Mol.Cell.Biol 5: 1639-1648; Hammer et. , 1987, Science 253: 53-58) ; the active α1- antitrypsin gene control region (Kelsey et al., In the liver, 1987, Genes and Devel 1: . 161-171); bone having activity in a cell-like a beta-globin gene control region (Mogram et al., 1985, Nature 315: 338-340; Kollias et al., 1986, Cell 46: 89-94); an active medulla in oligodendrocyte glial cells in the brain Phospholipid basic protein gene control region (Readhead et al., 1987, Cell 48: 703-712); a myosin light chain-2 gene control region active in skeletal muscle (Sani, 1985, Nature 314: 283-286) And an active gonadotropin releasing hormone gene control region in the hypothalamus (Mason et al., 1986, Science 234: 1372-1378).

An enhancer sequence can be inserted into a vector to increase transcription of a higher eukaryote against a DNA encoding an ST2 antigen binding protein of the invention comprising a light or heavy chain. Enhancers are cis-acting elements that act on a promoter to increase the transcription of DNA, typically from about 10-300 bp in length. The orientation and position of the enhancer are relatively independent and have been found at the 5' and 3' positions of the transcription unit. Several enhancer sequences are known to be available from mammalian genes (eg, globin, elastase, albumin, alpha-fetoprotein, and insulin). However, enhancers from viruses are often used. SV40 enhancers, cell giant viral early promoter enhancers, polyoma enhancers, and adenovirus enhancer lines are known in the art for use in activating such exemplary enhancers of eukaryotic promoters. Although the enhancer may be located in the vector 5' or 3' of the coding sequence, it is typically located at the 5' position of the promoter. A sequence encoding a suitable native or heterologous signal sequence (leader sequence or signal peptide) can be incorporated into a expression vector to facilitate extracellular secretion of the antibody. The choice of signal peptide or leader depends on the type of host cell in which the antibody is to be produced, and the heterologous signal sequence can be substituted for the native signal sequence. Examples of signal peptides that are functional in mammalian host cells include the following: interleukin-7 (IL-7) signal sequence, which is described in U.S. Patent No. 4,965,195; interleukin-2 receptor signal sequence, which is illustrated In Cosman et al., 1984, Nature 312:768; interleukin-4 receptor signal peptide, described in European Patent No. 0367 566; Type I interleukin-1 receptor signal peptide, described in the United States Patent No. 4,968,607; Type II interleukin-1 receptor signal peptide, which is described in European Patent No. 0 460 846.

The vector may contain one or more elements that promote expression when the vector is integrated into the host cell genome. Examples include EASE elements (Aldrich et al. 2003 Biotechnol Prog. 19:1433-38) and matrix attachment zone (MAR). MAR mediates the structural organization of chromatin and allows the integration vector to not produce a "position" effect. Therefore, MAR is especially useful when using vectors to generate stable transfectants. Numerous natural and synthetic MAR-containing nucleic acids are known in the art, for example, U.S. Patent Nos. 6,239,328, 7,326,567, 6,177,612, 6,388,066, 6,245,974, 7,259,010, 6,037,525, 7,422,874, 7,129,062.

The expression vector of the present invention can be constructed from a starting vector such as a commercially available vector. Such vectors may or may not contain all of the desired flanking sequences. In case one or more of the flanking sequences described herein If not already present in the vector, the flanking sequences are individually obtained and ligated to the vector. Methods for obtaining each of the flanking sequences are well known to those skilled in the art.

After the vector has been constructed and a nucleic acid molecule encoding an ST2 antigen-binding sequence comprising a light chain, a heavy chain or a light chain and a heavy chain has been inserted into a suitable site of the vector, the completed vector can be inserted into a suitable host cell for expansion. Increase and / or polypeptide performance. Expression vectors for ST2 antigen binding proteins can be transformed into selected host cells by conventional methods, including transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran Mediate transfection or other known techniques. The method chosen will vary to some extent depending on the type of host cell desired. Such methods and other suitable methods are well known to those skilled in the art and are described, for example, in Sambrook et al., 2001 (supra).

When cultured under suitable conditions, the host cell synthesizes an ST2 antigen-binding protein, which can then be derived from the culture medium (if the host cell secretes the ST2 antigen-binding protein into the culture medium) or directly from the host cell from which it was produced (if not The ST2 antigen binding protein is secreted and collected. The choice of a suitable host cell will depend on a variety of factors, such as the desired amount of expression, activity (e.g., glycosylation or phosphorylation), or the ease with which the desired polypeptide is modified and folded into a biologically active molecule. The host cell can be eukaryotic or prokaryotic.

Mammalian cell lines useful as expression hosts are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC) and can be used in any industry. The recombinant polypeptides of the invention are known to be used in cell lines of the expression system. In general, a host expression cell is transformed with a recombinant expression vector comprising a DNA encoding a desired anti-ST2 antibody polypeptide. Host cells that can be used are, in particular, prokaryotes, yeast or higher eukaryotes. Prokaryotes include Gram-negative or Gram-positive organisms such as E. coli or bacilli. Higher eukaryotic cells include insect cells and cell lines derived from mammals. Examples of suitable mammalian host cell lines include the COS-7 monkey kidney cell line (ATCC CRL 1651) (Gluzman et al, 1981, Cell 23: 175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL 163) Chinese hamster ovary (CHO) cells or their derivatives (eg Veggie CHO and related cell lines grown in serum-free medium) (Rasmussen et al., 1998, Cytotechnology 28:31), HeLa cells, BHK (ATCC CRL 10) Cell lines and CVI/EBNA cell lines derived from the African green monkey kidney cell line CVI (ATCC CCL 70) (as described by McMahan et al., 1991), EMBO J. 10: 2821, human embryonic kidney cells (eg 293, 293 EBNA or MSR 293), human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from nascent tissues, in vitro cultures of primary explants, HL -60, U937, HaK or Jurkat cells. Depending on the circumstances, for example, when it is desired to use the polypeptide in various signal transduction or reporter assays, mammalian cell lines such as HepG2/3B, KB, NIH 3T3 or S49 can be used to express the polypeptide. Alternatively, the polypeptide can be produced in a lower eukaryote such as yeast or in a prokaryote such as a bacterium. Suitable yeast include beer yeast (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Kluyveromyces (Kluyveromyces) strains, Candida (Candida) or capable of exhibiting a heterologous polypeptide of any yeast strain. Suitable strains include Escherichia coli, Bacillus subtilis , Salmonella typhimurium or any strain capable of expressing a heterologous polypeptide. If the polypeptide is made in yeast or bacteria, it may be desirable to modify the polypeptide produced therein by, for example, phosphorylation or glycosylation at a suitable site to obtain a functional polypeptide. Such covalent attachment can be achieved using known chemical or enzymatic methods. The polypeptide may also be operably linked to an appropriate control sequence and produced using an insect expression system by one or more insect expression vectors in one or more insect expression vectors. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, for example, Invitrogen, San Diego, Calif., USA (MaxBac® kits) and are well known in the art, such as Summers. And Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) and Luckow and Summers, Bio/Technology 6:47 (1988). The RNA derived from the nucleic acid constructs disclosed herein can also be produced using a cell-free translation system to produce the polypeptide. Suitable colonization and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Pouwels et al. ( Cloning Vectors: A Laboratory Manual , Elsevier, New York, 1985). A host cell line "recombinant host cell" comprising an isolated nucleic acid of the invention preferably operably linked to at least one expression control sequence is included.

In certain embodiments, cell lines can be selected by assaying cell lines that have high amounts of expression and that constitutively produce antigen-binding proteins with ST2 binding properties. In another embodiment, a B cell lineage selection cell line that has the ability to produce and secrete a heterologous antibody can be produced without the production of autoantibodies.

ST2 antigen binding protein of depleted cells

In a preferred embodiment, the ST2 antigen binding protein binds to ST2 and inhibits IL-33 binding, thereby reducing IL-33 mediated signaling in ST2 expressing cells. However, in certain embodiments, the ST2 antigen binding protein binds to ST2 and targets ST2 expressing cells for depletion. Of course, ST2 antigen binding proteins can inhibit IL-33 binding and target ST2 cells for depletion.

ST2 antigen binding proteins of depleted cells are particularly useful for treating diseases associated with ST2 overexpression, for example, inflammatory diseases or ST2 manifestations of tumors. Methods for targeting cells using antigen binding proteins (antibodies) are well known in the art. Example embodiments are discussed below.

Antibody drug conjugate

Embodiments of the invention include antibody drug conjugates (ADCs). Typically, the ADC comprises an antibody conjugated to a chemotherapeutic agent, eg, a cytotoxic agent, a cytostatic agent, a toxin or a radioactive agent. Linker molecules can be used to couple drugs to antibodies. Numerous types of linkers and drugs useful in ADC technology are known in the art and can be used in embodiments of the invention. (See US20090028856; US2009/0274713; US2007/0031402; WO2005/084390; WO2009/099728; US5208020; US5416064; US5475092; 5585499; 6436931; 6372738 and 6370071, all by reference The formula is incorporated herein).

Connector

In certain embodiments, the ADC comprises a linker comprised of one or more linker components. Exemplary linker components include 6-maleimido hexamethylene, maleimide propyl decyl, lysine- citrulline, alanine-phenylalanine, p-aminobenzyloxy The carbonyl group and the co-coupled with the linker reagent include, but are not limited to, N-succinimide 4-(2-succinimide) valerate ("SPP"), N-Amber醯imino 4-(N-maleimidomethyl)cyclohexane-1carboxylate ("SMCC", also referred to herein as "MCC") and N-amber quinone imine ( 4-iodo-ethenylamino benzoate ("SIAB").

The linker can be a "cleavable" linker or a "non-cleavable" linker (Ducry and Stump, Bioconjugate Chem. 2010, 21 , 5-13; the entire contents of which are incorporated herein by reference). The cleavable linker is designed to release the drug when subjected to certain environmental factors (eg, when internalized into the target cell). The cleavable linker comprises an acid labile linker, a protease sensitive linker, a photolabile linker, a dimethyl linker or a disulfide containing linker. After internalization by target cells and degradation in target cells, the non-cleavable linker tends to remain covalently associated with at least one amino acid of the antibody and the drug. An exemplary non-cleavable linkage system MCC.

drug

In certain embodiments, the anti-system is coupled to a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN.TM.); alkyl sulfonates such as busulfan and propylene sulfonate. Improsulfan) and piposulfan; aziridine, such as benzodopa, carboquone, meturedopa and uredopa; Imine and methyl melamine, including altretamine, tri-ethyl melamine, tri-ethylphosphoniumamine, tri-ethyl thiophosphonamide and trimethylol melamine; (acetogenin) (especially ballatacin and bullatacinone); camptothecin (including synthetic analogue topotecan); bryostatin; carris statins (callystatin); CC-1065 (including its Addo to new (adozelesin), to a new Kazhe (carzelesin) and the ratio of off to a new (bizelesin) synthetic analogs); cryptophycin (cryptophycin) (especially cryptalgal - 1 and cryptophycin 8); dolastatin (Dolastatin); Duokamixin (duocarmycin) (including synthetic analogs KW-2189 and CBI-TMI); Ai garnet plug Su (eleutherobin); H. littoralis base (pancratistatin); stolonifer coral alcohol (sarcodictyin); Spongistatin (spongistatin); nitrogen mustards, e.g. chlorambucil (chlorambucil), naphthalene nitrogen mustards (chlomaphazine), phosphorus chloride Cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, neonitrogen mustard (novembichin), phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosourea, such as carmustine Carmustine), chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine; antibiotics such as enediyne antibiotics For example, calicheamicin, especially calicheamicin.γ1 and calicheamicin θI, for example, see Angew Chem. Intl. Ed. Engl. 33: 183-186 (1994); Danamycin ( Dynemicin), including daantimycin A; esperamicin; and new Anti-cancer chromophore and related chromophorin diacetylene antibiotic chromophore), aclacinomysin, actinomycin, anthramycin, azoserine, Bora Bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin; chromomycin, dactinomycin, dao Daunorubicin, detorubicin, 6-diazo-5-oxo-L-positive leucine, doxorubicin (including morpholinyl- Dusalubicin, cyanomorpholinyl-dusabubicin, 2-pyrrolyl-dusalubicin and deoxyduzabubicin), epirubicin, Isobi Esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, culture Peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin ), streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate Meth (methotrexate) and 5-fluorouracil (5-FU); folic acid analogues such as dimethyl folate, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine Fludarabine, 6-巯嘌呤, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine (6- Azauridine), carmofur, cytarabine, dideoxyuridine, deoxyfluorouridine, enocitabine, fluorouridine, 5-FU; androgen, for example Calulsterone, dromostanolone propionate, epitiostolol, mepitiostane, azlactone; anti-adrenal, such as amine ubmet (aminoglutethimide), mitotane, trilostane; folic acid supplements such as folinic acid; vinegar Aceglatone; aldose glucoside; amino keto valeric acid; amsacrine; bestrabucil; bisantrene; edatrexate; Defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; epothilone; etoglucid ; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoid, such as maytansine and ansamitocin; mitoguazone; Mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; picric acid ; 2-ethyl hydrazine; procarbazine; PSK.RTM.; razoxane; rhizomycin; sizofuran; spirogermanium; tenuazonic acid ); triaziquone; 2,2',2"-trichlorotriethylamine; trichothecene (especially T-2) Toxin, verracurin A, roridin A, and anguidine; urethan; vindesine; dacarbazine ;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");cyclophosphamide;Shaotepa; taxoids such as paclitaxel (TAXOL.TM., Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (doxetaxel) (TAXOTERE.RTM., Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; guanidine; methotrexate; platinum analogues such as cisplatin Cisplatin) and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone醌;vincristine; vinorelbine; vinopenine (navelbine); able to kill (novantrone); teniposide (teniposide ;; daunomycin; aminylpterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethyl amnamine Acid (DMFO); retinoid; capecitabine; and any of the above pharmaceutically acceptable salts, acids or derivatives. Also included in this definition are anti-hormonal agents that modulate or inhibit the effects of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase inhibitory 4 (5) )-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Freis, Fareston); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; A pharmaceutically acceptable salt, acid or derivative as described above. Other chemotherapeutic agents that can be used with the present invention are disclosed in U.S. Publication No. 20080171040, or U.S. Patent Publication No. 20080305044, the entire disclosure of which is incorporated herein by reference.

It is contemplated that the antibody can be coupled to two or more different chemotherapeutic agents, or the pharmaceutical composition can comprise an antibody mixture wherein the antibody components are the same but coupled to different chemotherapeutic agents. These embodiments can be used to target a variety of biological pathways of target cells.

In a preferred embodiment, the ADC comprises an antibody coupled to one or more class of maytansine molecules that act by inhibiting tubulin polymerization to act as a mitotic inhibitor. Maytans (including various modifications) are described in the following patents: U.S. Patent Nos. 3,896,111, 4,151,042, 4,137,230, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, No. 4307016, No. 4308268, No. 4309428, No. 4313946, No. 4315929, No. 4317821, No. 4322348, No. 4331598, No. 4361650, No. 4364866, No. 4424219, No. 4450254, No. 4362663 No., No. 4371533 and WO 2009/099728. The cantansinoid moiety can be isolated from natural sources, produced using recombinant techniques or prepared synthetically. Exemplary cantansins include C-19-dechlorination (U.S. Patent No. 4,256,746), C-20-hydroxy (or C-20-demethylation) +/- C-19-dechlorination (US Patent No. 4,370,016) No. 4,361,650), C-20-desmethoxy (or C-20-decyloxy) (-OCOR), +/- dechlorination (US Patent No. 4294757), C-9-SH (USA) Patent No. 4,424,219), C-14-alkoxymethyl (demethoxy/CH2OR) (U.S. Patent No. 4,331,598), C-14-hydroxymethyl or decyloxymethyl (CH2OH or CH2OAc) ( U.S. Patent No. 4,450,254), C-15-hydroxy/decyloxy (U.S. Patent No. 4,364,866), C-15-methoxy (US Patent No. 4,313,946) No. 4,315, 533), C-18-N-demethylation (U.S. Patent Nos. 4,362,663 and 4,322,348) and 4,5-deoxygenation (U.S. Patent No. 4,371,533).

Different positions on the maytansinoid compound can be used as the attachment position, depending on the type of linkage desired. For example, in the formation of an ester linkage, the C-3 position having a hydroxyl group, the C-14 position modified by a hydroxymethyl group, the C-15 position modified by a hydroxyl group, and the C-20 position having a hydroxyl group are all suitable (US Patent) U.S. Patent Application Publication No. 20050167245 and No. 20070037972 and WO 2009099728).

Preferred maytansoids include those which are referred to in the art as DM1, DM3, and DM4 (U.S. Patent Application Publication Nos. 2009030924 and No. 20050276812, each of which is incorporated herein by reference).

ADCs containing melamine-like, methods of preparing such ADCs, and therapeutic applications thereof are disclosed in U.S. Patent Nos. 5,208,020 and 5, 516, 640, U.S. Patent Application Publication No. 20050276812, and WO 2009099728, all incorporated herein by reference. )in. Linkers useful for the preparation of memantine-like ADCs are known in the art (U.S. Patent No. 5,208,020 and U.S. Patent Application Publication Nos. 2005016993 and No. 20090274713; herein incorporated by reference in their entirety). A cantanal ADC comprising a SMCC linker can be prepared as disclosed in U.S. Patent Publication No. 2005/0276812.

Effector function enhancing antibody

One function of the Fc portion of an antibody is to communicate with the immune system when the antibody binds to its target. This is considered an "effect sub-function." Communication results in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cellular cytotoxicity (CDC). ADCC and ADCP are mediated via binding of Fc to Fc receptors on the surface of immune system cells. CDC is mediated via Fc binding to proteins of the complement system (eg, Clq).

IgG subclasses differ in their ability to mediate effector functions. For example, IgG1 is far superior to IgG2 and IgG4 in mediating ADCC and CDC. Therefore, in the performance of destruction ST2 In the examples in which the cells are targets, the anti-ST2 IgG1 antibody is preferred.

The effector function of the antibody can be increased or decreased by introducing one or more mutations into the Fc. Embodiments of the invention include antigen binding proteins having Fc engineered to increase effector function, for example, antibodies (US 7,317,091 and Strohl, Curr . Opin . Biotech., 20:685-691, 2009; both Both are incorporated herein by reference). Exemplary IgGl Fc molecules with increased effector function include (based on the Kabat numbering scheme) they have the following substitutions: S239D/I332E

S239D/A330S/I332E

S239D/A330L/I332E

S298A/D333A/K334A

P247I/A339D

P247I/A339Q

D280H/K290S

D280H/K290S/S298D

D280H/K290S/S298V

F243L/R292P/Y300L

F243L/R292P/Y300L/P396L

F243L/R292P/Y300L/V305I/P396L

G236A/S239D/I332E

K326A/E333A

K326W/E333S

K290E/S298G/T299A

K290N/S298G/T299A

K290E/S298G/T299A/K326E

K290N/S298G/T299A/K326E.

Other embodiments of the invention include Fc having modifications to reduce effector function Antigen binding proteins, for example, antibodies. Exemplary Fc molecules with reduced effector functions include (based on the Kabat numbering scheme) which have the following substitutions: N297A (IgGl)

L234A/L235A (IgG1)

V234A/G237A (IgG2)

L235A/G237A/E318A (IgG4)

H268Q/V309L/A330S/A331S (IgG2)

C220S/C226S/C229S/P238S (IgG1)

C226S/C229S/E233P/L234V/L235A (IgG1)

L234F/L235E/P331S (IgG1)

S267E/L328F (IgG1).

Another way to increase the effector function of IgG-containing Fc proteins is by reducing the fucosylation of Fc. Removal of nuclear fucose from the biantennary complex oligosaccharide attached to Fc significantly increases ADCC effector function without altering antigen binding or CDC effector function. Several ways to reduce or eliminate fucosylation of Fc-containing molecules (eg, antibodies) are known. These include recombinant expression in certain mammalian cell lines, including FUT8 knockout cell lines, variant CHO line Lec13, rat hybridoma cell line YB2/0, including specifically targeting FUT8 A cell line of small interfering RNA of a gene and a cell line which together express β-1,4- N -ethyl glucosyltransferase III and Golgi α mannosidase II. Alternatively, the Fc-containing molecule can be expressed in non-mammalian cells (eg, plant cells, yeast, or prokaryotic cells (eg, E. coli)). Thus, in certain embodiments of the invention, the composition comprises an antibody having reduced fucosylation or complete afucosylation, eg, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10 or Ab11.

Pharmaceutical composition

In some embodiments, the invention provides a pharmaceutical composition comprising a therapeutically effective amount One or more of the antigen-binding proteins of the invention and pharmaceutically effective diluents, carriers, solubilizers, emulsifiers, preservatives and/or adjuvants. In certain embodiments, the antigen binds to a protein-based antibody. Pharmaceutical compositions of the invention include, but are not limited to, liquid, frozen, and lyophilized compositions.

Preferably, the formulation material is non-toxic to the recipient at the dosages and concentrations employed. In a particular embodiment, a pharmaceutical composition is provided comprising a therapeutically effective amount of an ST2 antigen binding protein, such as an ST2 binding antibody.

In certain embodiments, a pharmaceutical composition may contain a formulation material for modifying, maintaining, or maintaining, for example, the following properties of the composition: pH, volumetric osmolality, viscosity, clarity, color, isotonicity, odor, The rate of sterility, stability, dissolution or release, absorption or penetration. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (eg, glycine, glutamic acid, aspartame, arginine, valine or lysine); antimicrobial An antioxidant (such as ascorbic acid, sodium sulfite or sodium bisulfite); a buffer (such as borate, bicarbonate, Tris-HCl, citrate, phosphate or other organic acid); a swelling agent (such as mannitol or Glycine; chelating agent (eg ethylenediaminetetraacetic acid, EDTA); complexing agent (eg caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin); filler Monosaccharide; disaccharide; and other carbohydrates (such as glucose, mannose or dextrin); protein (such as serum albumin, gelatin or immunoglobulin); colorants, flavors and diluents; emulsifiers; (eg, polyvinylpyrrolidone); low molecular weight polypeptide; salt forming counterion (eg, sodium); preservative (eg, benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol) , methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, Chlorhexidine, sorbic acid or hydrogen peroxide; solvent (eg glycerol, propylene glycol or polyethylene glycol); sugar alcohol (eg mannitol or sorbitol); suspending agent; surfactant or wetting agent (eg, pluronic, PEG, sorbitan ester, polysorbate (eg, polysorbate 20, polysorbate), triton, tromethamine, Lecithin, cholesterol, tyloxapal; stability enhancers (eg sucrose or sorbitol); tonicity enhancers (eg alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbus) Alcohol); delivery vehicle; diluent; excipient and/or pharmaceutical adjuvant. See REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed. (edited by A.R. Genrmo), 1990, Mack Publishing.

In certain embodiments, the optimal pharmaceutical composition can be determined by those skilled in the art based on, for example, established routes of administration, mode of delivery, and desired dosage. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, see above. In certain embodiments, such compositions can affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the antigen binding proteins of the invention. In certain embodiments, the initial vehicle or carrier in the pharmaceutical composition can be substantially aqueous or non-aqueous. For example, a suitable vehicle or carrier can be water for injection, physiological saline or artificial cerebrospinal fluid, which may be supplemented with other common materials in parenteral compositions. Neutral buffered saline or saline mixed with serum albumin is another exemplary vehicle. In a particular embodiment, the pharmaceutical composition comprises a Tris buffer having a pH of about 7.0-8.5 or an acetate buffer having a pH of about 4.0-5.5, and may further comprise sorbitol or a suitable replacement thereof. In certain embodiments of the invention, an ST2 antigen binding protein composition (REMINGTON'S PHARMACEUTICAL) in the form of a lyophilized cake or aqueous solution for storage can be prepared by mixing selected compositions of the desired purity with an optional formulation. SCIENCES, see above). Moreover, in certain embodiments, the ST2 antigen binding protein product can be formulated as a lyophilizate using suitable excipients such as sucrose.

The pharmaceutical compositions of the invention may be selected for parenteral delivery. Alternatively, the composition can be selected for inhalation or for delivery by the digestive tract, such as by oral delivery. The preparation of such pharmaceutically acceptable compositions is well known to those skilled in the art. The formulation component is preferably present at a concentration that is acceptable for the site of administration. In certain embodiments, the composition is maintained at a physiological pH or a slightly lower pH using a buffer, typically in a pH range of from about 5 to about 8.

When administered parenterally, the therapeutic compositions useful in the present invention may be provided in the form of a non-pyrogenic, parenterally acceptable aqueous solution comprising the desired ST2 antigen binding in a pharmaceutically acceptable vehicle. protein. A vehicle suitable for parenteral injection is sterile distilled water in which the ST2 antigen binding protein is formulated into a sterile isotonic solution and stored in an appropriate manner. In certain embodiments, the preparation may involve the formulation of a desired molecule with an agent that allows controlled release or sustained release of the product, such as injectable microspheres, bioerodible particles, polymeric compounds (eg, polylactic acid or polyglycolic acid), beads. Granules or liposomes, which can be delivered by depot injection. In certain embodiments, hyaluronic acid can also be used, which has the effect of prolonging the duration of the cycle. In certain embodiments, an implantable drug delivery device can be used to introduce a desired antigen binding protein.

The pharmaceutical composition of the invention may be formulated for inhalation. In these embodiments, the ST2 antigen binding protein is advantageously formulated as a dry inhalable powder. In a particular embodiment, the ST2 antigen binding protein inhalation solution can also be formulated with a propellant for aerosol delivery. In certain embodiments, the solution can be sprayed. Thus, the pulmonary administration and formulation method is further described in International Patent Application No. PCT/US94/001875, the disclosure of which is incorporated herein by reference in its entirety in its entirety.

It is also contemplated that the formulation can be administered orally. The ST2 antigen binding protein administered in this manner can be formulated in the presence or absence of a carrier that is typically used to complex solid dosage forms such as tablets and capsules. In certain embodiments, the capsule can be designed to release the active portion of the formulation at a site in the gastrointestinal tract to maximize bioavailability and minimize systemic pre-degradation. Other agents may be included to facilitate absorption of the ST2 antigen binding protein. Diluents, flavoring agents, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binding agents can also be employed.

Other pharmaceutical compositions, including formulations involving sustained delivery or controlled delivery of the formulation involving the ST2 antigen binding protein, will be apparent to those skilled in the art. Surgery for the deployment of a variety of other continuous delivery or controlled delivery methods, such as liposome carriers, bioecution The disintegration of microparticles or porous beads and the accumulation of injections are also known to those skilled in the art. See, for example, International Patent Application No. PCT/US93/00829, which is incorporated herein incorporated by reference in its entirety in its entirety in its entirety in the the the the the the the the the Sustained release formulations may include a semipermeable polymeric matrix, such as a film or microcapsule, in the form of a shaped article. Sustained release matrices may include polyesters, hydrogels, polylactides (as disclosed in U.S. Patent No. 3,773,919 and European Patent Application Publication No. EP 058481, each of which is incorporated by reference herein) Copolymer of L-glutamic acid-γ-ethyl ester (Sidman et al., 1983, Biopolymers 2: 547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15: 167-277; and Wrangell, 1982, Chem. Tech. 12: 98-105), ethylene vinyl acetate (Langer et al., 1981, supra) or poly D (- -3-hydroxybutyric acid (European Patent Application Publication No. EP 133,988). Sustained release compositions can also include liposomes that can be prepared by any of a number of methods known in the art. See, for example, Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82: 3688-3692; European Patent Application Publication No. EP 036,676; EP 088, 046, and EP 143,949, incorporated herein by reference. .

Pharmaceutical compositions for in vivo administration are usually provided in the form of a sterile preparation. Sterilization can be carried out by filtration through a sterile filter. Sterilization using this method can be carried out before or after lyophilization and reconstitution when the composition is lyophilized. Compositions for parenteral administration can be stored in lyophilized form or in solution. The parenteral compositions are typically placed in a container having a sterile access port (e.g., an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle).

The present invention includes a self-buffering ST2 antigen-binding protein formulation, which can be used as a pharmaceutical composition, as described in International Patent Application No. WO 06138181 A2 (PCT/US2006/022599), the entire contents of which is incorporated by reference. Incorporated herein.

As discussed above, certain embodiments provide ST2 antigen binding protein compositions, particularly The pharmaceutical ST2 antigen binding protein composition further comprises one or more excipients in addition to the ST2 antigen binding protein, such as those described in this section and elsewhere herein. Excipients can be used in the present invention for a variety of purposes in this regard, for example, to adjust the physical, chemical or biological properties of the formulation, such as to adjust viscosity; and or to be used in the methods of the invention to improve effectiveness and or to stabilize Isoforms and methods for combating degradation and spoilage caused by, for example, stresses occurring during, during, and after preparation, delivery, storage, pre-use, and during administration.

A variety of explanations regarding protein stabilization and formulation materials and methods useful in this regard can be found, for example, in Arakawa et al., "Solvent interactions in pharmaceutical formulations", Pharm Res. 8(3): 285-91 (1991); Kendrick et al. "Physical stabilization of proteins in aqueous solution", RATIONAL DESIGN OF STABLE PROTEIN FORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning, ed., Pharmaceutical Biotechnology. 13: 61-84 (2002) and Randolph et al., "Surfactant-protein interactions", Pharm Biotechnol. 13: 159-75 (2002), the entire contents of each of which are hereby incorporated by reference, in particular in connection with the present disclosure of the present invention and its methods for self-buffering protein formulations Part of this, especially with regard to protein medicinal products and methods for veterinary and/or human medical applications.

Salts can be used, for example, to adjust the ionic strength and/or isotonicity of the formulation and/or to improve the solubility and/or physical stability of the protein or other component of the compositions of the present invention in accordance with certain embodiments of the present invention. .

As is well known, ions can stabilize the natural state of a protein by binding charged residues on the surface of the protein and blocking charged and polar groups in the protein and reducing the intensity of their electrostatic interactions, attractive and repulsive interactions. . Ions can also stabilize protein denaturation by binding to a specific protein-denatured peptide linkage (--CONH). state. In addition, ionic interactions with charged and polar groups in proteins can also reduce intermolecular electrostatic interactions and thereby prevent or reduce protein aggregation and insolubility.

Ionic species differ significantly in their effect on proteins. A number of ion classifications and their effects on proteins have been developed which can be used to formulate pharmaceutical compositions of the invention. One example is the Hofmeister series, which classifies ions and polar nonionic solutes by the effect of conformational stabilization of proteins in solution. A stable solute is called a "kosmotropic". Destabilizing solutes is called "chaotropic". The lyophilic agent is typically used at a high concentration (e.g., > 1 molar concentration of ammonium sulfate) to precipitate protein from the solution ("salting out"). Chaotropic agents are commonly used to denature and/or dissolve proteins ("salting-in"). The relative validity of ions to "salt-in" and "salting out" defines their position in the Hofmeister series.

Free amine groups can be used in ST2 antigen binding protein formulations in accordance with various embodiments of the invention as bulking agents, stabilizers, and antioxidants, as well as other standard applications. Amino acids, proline, serine and alanine can be used to stabilize the protein in the formulation. Glycine can be used in lyophilization to ensure accurate cake structure and properties. Arginine can be used to inhibit protein aggregation in both liquid and lyophilized formulations. Methamine can be used as an antioxidant.

Polyols include sugars such as mannitol, sucrose, and sorbitol; and polyhydric alcohols such as glycerin and propylene glycol; and polyethylene glycol (PEG) and related materials for the purposes of the discussion herein. Polyol is a lyophile. It is a useful stabilizer in both liquid and lyophilized formulations to protect proteins from physical and chemical degradation processes. Polyols can also be used to adjust the tension of the formulation.

Polyols which may be used to select embodiments of the invention are, in particular, mannitol, which are generally used to ensure the structural stability of the cake in the lyophilized formulation. It ensures the structural stability of the cake. It is usually used with a lyoprotectant (eg, sucrose). Sorbitol and sucrose are preferred reagents for adjusting tension during bulk material handling or preparation during the manufacturing process and as a stabilizer to resist freeze-thaw stress. Such as glucose and lactose Raw sugars, which contain free aldehyde or ketone groups, saccharify the surface from the amine and arginine residues. Therefore, it is generally not a particularly preferred polyol for use in the present invention. Further, the sugar (e.g., sucrose) forming these reactive substances is hydrolyzed to fructose and glucose under acidic conditions and then causes saccharification, and in this regard, it is not a particularly preferred polyol of the present invention. PEG can be used to stabilize proteins and as a lyoprotectant and is useful in the present invention.

Examples of ST2 antigen binding protein formulations further comprise a surfactant. Protein molecules can readily adsorb on surfaces and are susceptible to denaturation and subsequent aggregation at the air-liquid, solid-liquid, and liquid-liquid interfaces. The magnitude of these effects is usually inversely proportional to the protein concentration. The scale of such deleterious interactions is generally inversely proportional to the protein concentration and is often exacerbated by physical agitation (eg, during generation during product delivery and disposal).

Surfactants are commonly used to prevent, minimize or reduce surface adsorption. In this regard, surfactants useful in the present invention include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylate, and poloxamer 188.

Surfactants are also commonly used to control protein conformational stability. The use of surfactants in this regard is protein specific, as any given surfactant will generally stabilize some proteins and destabilize others.

Polysorbates are susceptible to oxidative degradation and are usually supplied with a sufficient amount of peroxide to cause oxidation of the side chains of the protein residues, especially methionine. Therefore, polysorbates should be used with care and, at the time of use, at their lowest effective concentration. In this regard, polysorbates exemplify the general rule that excipients should be used at their lowest effective concentration.

Examples of ST2 antigen binding protein formulations further comprise one or more antioxidants. Harmful oxidation of proteins in pharmaceutical formulations can be prevented by maintaining an appropriate amount of ambient oxygen and temperature and by avoiding exposure to light to some extent. Antioxidant excipients can also be used to prevent oxidative degradation of proteins. In this regard, antioxidant-based reducing agents, oxygen/radical scavengers, and chelating agents are especially useful. For use in the therapeutic protein formulations of the invention The antioxidant is preferably water soluble and maintains its activity throughout the shelf life of the product. In this regard, EDTA is a preferred antioxidant of the present invention.

Antioxidants can damage proteins. For example, reducing agents such as glutathione can disrupt intramolecular disulfide linkages. Accordingly, the antioxidants useful in the present invention are selected to specifically eliminate or substantially reduce the likelihood that the protein itself will be compromised.

Formulations of the invention may include a metal cofactor as a protein cofactor and are required to form a protein coordination complex, such as zinc required to form certain insulin suspensions. Metal ions can also inhibit some of the process of degrading proteins. However, metal ions also catalyze the physical and chemical processes that degrade proteins.

Magnesium ions (10-120 mM) can be used to inhibit the isomerization of aspartic acid to isoaspartic acid. Ca ⁺² ions (up to 100 mM) increase the stability of human DNase. However, Mg ⁺² , Mn ⁺² and Zn ⁺² can destabilize rhDNase. Similarly, Ca ⁺² and Sr ⁺² stabilize the factor VIII, Mg ⁺² , Mn ⁺² and Zn ⁺² , Cu ⁺² and Fe ⁺² to destabilize it, and Al ⁺³ ion can increase its aggregation.

Examples of ST2 antigen binding protein formulations further comprise one or more preservatives. Preservatives are required when developing involves multiple doses of parenteral formulations that are extracted more than once from the same container. Its primary function is to inhibit microbial growth and ensure product sterility throughout the shelf life or lifetime of the drug product. Commonly used preservatives include benzyl alcohol, phenol and m-cresol. Although preservatives have a long history of use as small molecule parenteral materials, the development of protein formulations including preservatives can be challenging. Preservatives almost always have a destabilizing effect (aggregation) on proteins, and this has become a major factor limiting their use in multi-dose protein formulations. To date, most protein drugs have been formulated for single use. However, when a multi-dose formulation is achievable, it has the added advantage of being convenient for the patient and increases the marketability. A good example is a preservative for human growth hormone (hGH), where the development of antiseptic formulations has led to the commercialization of more convenient, multi-purpose pen presentations. At least 4 such pen devices containing hGH antiseptic formulations are currently available in the market. Got it. Nordictropin (Liquid, Novo Nordisk), Nutropin AQ (Liquor, Genentech) and Genotropin (Freeze-Dual Chamber, Pharmacia & Upjohn) Phenol, while Somatrope (Eli Lilly) was formulated with m-cresol.

Several aspects need to be considered during preservative formulation and development. The effective preservative concentration in pharmaceutical products must be optimized. There is a need to test a given preservative in a dosage form in a concentration range that imparts antimicrobial effectiveness without compromising protein stability.

It is expected that the development of liquid formulations containing preservatives is more challenging than lyophilized formulations. The lyophilized product can be lyophilized without the use of a preservative and reconstituted with a preservative-containing diluent at the time of use. This shortens the time that the preservative is in contact with the protein, thereby significantly minimizing the associated stability risk. For liquid formulations, preservative effectiveness and stability should be maintained throughout the shelf life (approximately 18 to 24 months). It is important to note that the final formulation containing the active drug and all excipient components should show the effectiveness of the preservative.

ST2 antigen binding protein formulations are typically designed for specific administration routes and methods, particularly for specific administration doses and administration frequencies, for specific treatments for a particular disease, particularly in terms of certain bioavailability and persistence. Thus, formulations may be designed according to the present invention for delivery by any suitable route including, but not limited to, oral, otic, ocular, rectal, and vaginal and by parenteral routes, including intravenous and intra-arterial injections. Intramuscular injection and subcutaneous injection.

After the pharmaceutical composition has been formulated, it can be stored as a solution, suspension, gel, emulsion, solid, crystal or as a dehydrated or lyophilized powder in a sterile vial. Such formulations may be stored in ready-to-use form or in a form that is reconstituted prior to administration (eg, lyophilized). The invention also provides kits for producing single dose administration units. Each of the kits of the present invention can contain both a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments of the invention, a single chamber and multi-chamber pre-filled syringe is provided (eg, a liquid injection) The set of ejector and lysyringe injections.

The therapeutically effective amount of the pharmaceutical composition to be employed containing the ST2 antigen binding protein will depend, for example, on the therapeutic background and objectives. Those skilled in the art will appreciate that the appropriate dosage amount for treatment will vary, depending in part on the molecule being delivered, the indication for which the ST2 antigen binding protein is used, the route of administration, and the size of the patient (weight, body surface or Organ size) and/or condition (age and general health). In certain embodiments, the clinician can titrate the dose and modify the route of administration to achieve optimal therapeutic effects. Typical dosages can range from about 0.1 [mu]g/kg to up to about 30 mg/kg or higher, depending on the above factors. In particular embodiments, the dosage may range from 1.0 [mu]g/kg to up to about 20 mg/kg, optionally in the range of from 10 [mu]g/kg to up to about 10 mg/kg or from 100 [mu]g/kg up to about 5 mg/kg. Inside.

A therapeutically effective amount of the ST2 antigen binding protein preferably reduces the severity of the symptoms of the disease, increases the frequency or duration of the disease-free symptom period, or prevents injury or disability due to susceptibility.

The pharmaceutical composition can be administered using a medical device. Examples of medical devices for administering pharmaceutical compositions are described in U.S. Patent Nos. 4,475,196, 4,439,196, 4,447,224, 4,447,233, 4,486,194, 4,487,603, 4,596,556, 4,790,824. , 4,941, 880, 5, 064, 413, 5, 312, 335, 5, 312, 335, 5, 383, 851, and 5, 399, 163, each of which is incorporated herein by reference.

Method of diagnosing or treating an ST2-related disease or condition

The ST2 antigen binding protein of the invention is particularly useful for detecting ST2 in biological samples. In certain embodiments, a biological sample obtained from a patient is contacted with an ST2 antigen binding protein. The binding of the ST2 antigen binding protein to ST2 is then tested to determine the presence or relative amount of ST2 in the sample. These methods can be used to diagnose or determine a patient suitable for treatment with an ST2 antigen binding protein.

In certain embodiments, the ST2 antigen binding proteins of the invention are used to diagnose, detect or treat autoimmune or inflammatory conditions. In the treatment of autoimmune or inflammatory conditions, ST2 antigen binding proteins can target ST2 expression cells of the immune system for disruption and/or can block the interaction of ST2 with IL-33.

Conditions associated with IL-33 mediated signaling are particularly suitable for treatment with one or more of the ST2 antigen binding proteins disclosed herein. Such conditions include, but are not limited to, inflammation, autoimmune diseases, tumor-associated autoimmune diseases, cartilage inflammation, fibrotic diseases and/or bone degradation, arthritis, rheumatoid arthritis, juvenile arthritis , juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile type of sexual spondylitis, juvenile Enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's Syndrome, SEA syndrome (serum-negative, bone-point lesions, joint disorders), juvenile dermatomyositis, juvenile psoriasis Arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, adhesion Spondylitis, enteropathic arthritis, reactive arthritis, Respiratory syndrome, SEA syndrome (serum-negative, bone-point lesions, joint disorders), dermatomyositis, psoriasis Arthritis, scleroderma, systemic lupus erythematosus, vasculitis, myositis, polymyositis, dermatomyositis, nodular polyarteritis, Wagner's granuloma, arteritis, rheumatic polymyalgia, Sarcoma, scleroderma, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, psoriasis, pleated psoriasis, pustular Psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD) , Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, sinusitis, sinusitis with polyps, eosinophilic esophagitis, eosinophilia Tracheitis Guillain-Barre disease, type 1 diabetes, thyroiditis (eg, Graves' disease), Addison's disease, Raynaud's phenomenon, autologous Immunological hepatitis, GVHD, transplant rejection, kidney damage, and the like.

In a preferred embodiment, the autoimmune or inflammatory condition is asthma, atopic dermatitis, chronic obstructive pulmonary disease, pulmonary fibrosis, sepsis and trauma, HIV infection, systemic lupus erythematosus, inflammatory bowel disease, rheumatoid Arthritis, cirrhosis, Wagner's granulomatosis, Beth's disease, cardiovascular disease, sinusitis, nasal polyps and eosinophilic bronchitis.

In certain embodiments, the ST2 antigen binding proteins of the invention are used to diagnose, detect or treat a cancer or tumorigenic disorder. In the treatment of cancer or tumorigenic disorders, the ST2 antigen binding protein can target ST2 expressing cells for disruption and/or can block the interaction of ST2 with IL-33, thereby reducing IL-33 mediated signaling. . For example, the performance of highly soluble ST2 is associated with improved survival in breast cancer patients. (Prechtel et al, Lab Invest (2001) 81: 159-165). Since soluble ST2 binds and blocks IL-33 mediated signaling, it is expected that ST2 antigen binding proteins that block IL-33 mediated signaling can be used to promote improved survival in breast cancer patients. Cancer or tumorigenic disorders that can be diagnosed, detected, or treated with ST2 antigen binding proteins include, but are not limited to, solid tumors, typically lung cancer, ovarian cancer, breast cancer, prostate cancer, endometrial cancer, kidney cancer, esophageal cancer, pancreas Dirty cancer, squamous cell carcinoma, uveal melanoma, cervical cancer, colorectal cancer, bladder cancer, brain cancer, pancreatic cancer, head cancer, cervical cancer, liver cancer, leukemia, lymphoma and Hodgkin's disease (Hodgkin's disease), multiple myeloma, melanoma, gastric cancer, stellate glial carcinoma, stomach and lung adenocarcinoma.

Antigen binding proteins can be used to inhibit tumor growth, progression and/or metastasis. This inhibition can be monitored using a variety of methods. For example, inhibition can reduce tumor size and/or reduce metabolic activity within the tumor. These parameters can all be measured by, for example, MRI or PET scanning. Inhibition can also be monitored by biopsy to determine the degree of necrosis, tumor cell death and vascular distribution in the tumor. Degree of vascularity. The degree of transfer can be monitored using known methods.

Instance

The following examples (both actual and predicted) are provided for the purpose of illustrating the specific embodiments or features of the invention and are not intended to limit the scope thereof.

Example 1: ST2 antibody is effective in an animal model of asthma

This example demonstrates that administration of antibodies that bind to ST2 and inhibit IL-33 mediated signaling is effective in an animal model of inflammatory disease (ie, asthma). Neutralizing mouse ST2 mAb (ST2 substitute mAb) inhibited the activity of exogenously administered IL-33 in vivo. Two hundred hours after intravenous injection of 100 ug of anti-ST2 mAb, 200 ng of recombinant mouse IL-33 was administered intranasally to mice. The next day, the IL-5 concentration of the branched tracheal alveolar lavage fluid (BALF) was measured by ELISA. Baseline IL-5 concentrations were obtained from BALF of saline treated mice prior to saline stimulation. The maximal BALF IL-5 concentration was obtained from mice treated with IL-33 stimulated isotype control Ig. ST2 mAb treatment significantly inhibited IL-33-induced IL-5 in BALF of BALB/c and C57BL/6 mouse strains compared to isotype control Ig treatment (Figure 1).

The ST2 substitute mAb was effective in a sputum allergen (CRA)-induced asthma model in which BALF eosinophils were significantly less in ST2 antibody-treated mice than in isotype control Ig-treated mice. On day 1, day 3, day 6, day 8, day 10 and day 13, BALB/c mice were stimulated with 100 [mu]g CRA. On day 0, day 7, and day 13, mice were injected with 250 [mu]g of anti-ST2 mAb or isotype control Ig, with antibody injection on day 13 prior to final intranasal CRA stimulation. On day 14, the mice were anesthetized and subjected to lung lavage. Counting the number of BALF cell populations and treatment with anti-ST2 mAb resulted in the presence of significantly fewer total BALF cells, with eosinophils comprising a significantly affected population of cells (Figure 2).

Example 2: Generation of anti-ST2 antibodies using the Xenomouse® platform

Fully human antibodies to human ST2 are generated using the XENOMOUSE® technique (U.S. Patent Nos. 6,114,598, 6, 162, 963, 6, 833, 268, 7, 049, 426, 7, 064, 244, the entire contents of each of 1994, Nature Genetics 7: 13-21; Mendez et al., 1997, Nature Genetics 15: 146-156; Green and Jakobovitis, 1998, J. Ex. Med. 188: 483-495, Kellermann and Green, 2002, Current Opinion In Biotechnology , 13: 593-597).

XMG2K, XMG4K and XMG4KL XENOMOUSE® animals were immunized with a polypeptide comprising the extracellular domain of human ST2 fused to the human antibody Fc domain or with a complex of human ST2-Fc fusion protein and human IL-33. The initial immunization of XENOMOUSE® animals was carried out according to the method disclosed in the following cases using a suitable amount of immunogen (i.e., 10 [mu]g of soluble ST2/mouse): U.S. Patent Application Serial No. 08/759,620 and 1998, filed on December 3, 1996. The international patent application No. WO 98/24893, published on Jun. 11, 2011, and WO 00/76310, issued on Dec. 21, 2000, the disclosure of which is incorporated herein by reference. Following initial immunization, subsequent booster immunization of the immunogen (5 [mu]g soluble ST2 or ST2/IL33 complex/mouse) was administered as planned and the duration required to induce the appropriate potency of the anti-ST2 antibody in mice was maintained. The titer is determined by a suitable method (e.g., ELISA or fluorescent activated cell sorting (FAC)).

Animals exhibiting appropriate potency were identified and lymphocytes were obtained from draining lymph nodes and pooled for each group as needed. Lymphatic tissue is ground in a suitable medium (eg, Dulbecco's Modified Eagle Medium; DMEM; available from Invitrogen, Carlsbad, CA) to separate from the lymphocytes to allow the tissue to release cells and Suspended in DMEM. B cells are selected and/or expanded using suitable methods and are suitably fused to a subject using techniques known in the art (eg, non-secretory myeloma P3X63Ag8.653 cells (American Type Culture Collection CRL 1580; Kearney et al, J. Immunol. 123, 1979, 1548-1550)) Fusion.

In a fusion method, lymphocytes are mixed with cells of fusion target at a ratio of 1:4. The cell mixture was slowly pelleted by centrifugation at 400 xg for 4 minutes, the supernatant was decanted, and the cell mixture was gently mixed (eg, by using 1 mL pipetting) tube). Fusion was induced with PEG/DMSO (polyethylene glycol/dimethylammonium; available from Sigma-Aldrich, St. Louis MO; 1 mL/million lymphocytes). While gently agitating, PEG/DMSO was slowly added over 1 minute, followed by mixing for 1 minute. IDMEM (DMEM, gluten-free valine; 2 mL/million B cells) was then added over 2 minutes with gentle agitation, followed by the addition of additional IDMEM (8 mL/million B cells) over 3 minutes.

The fused cells were slowly pelleted (400 x g for 6 minutes) and resuspended in 20 mL of selection medium (DMEM containing azosergic acid and hypoxanthine [HA] and other supplemental materials as needed) / million In B cells. The cells were incubated at 37 °C for 20-30 minutes and then resuspended in 200 mL of selection medium and cultured in T175 flasks for 3 to 4 days and then plated in 96-well plates.

Cells were dispensed into 96-well plates using standard techniques to maximize the degree of colony formation. After several days of culture, the supernatant was collected and subjected to screening analysis. Hybridoma supernatants generated from mice immunized with the ST2-Fc/IL33 complex were screened by ELISA-based assay using 0.5 ug/mL ST2-Flag/his and human IL-33 The complex was passively coated overnight at 96 °C with a 96-well polystyrene ELISA plate. For the determination of ST-2 specific binding, a second ELISA screen was performed using a 96-well polystyrene plate that was passively coated overnight at 10 °/mL with neutral neutravidin at 4 °C. It was then washed with 0.5 ug/mL biotinylated human IL33 and loaded onto an assay plate. This ELISA screening method can identify more than 1,200 anti-ST2 specific binding agents.

ST2 antigen-specific antibodies from hybridoma supernatants produced by soluble ST2-Fc immunized mice were screened by fluorescence assay microfluidic analysis (FMAT) for transient transfection of full-length human ST2 Recombinant HEK293T cells were screened and counter-screening was performed against mock transfected HEK293T cells. Briefly, cells were seeded into 384-well FMAT plates at a density of 40 ul/well in accordance with the density of 6,000 ST2 positive cells/well and 14,000 mock transfected ST2 negative cells/well. The hybridoma supernatant was then added and allowed to bind for 1 hour at room temperature, followed by washing and use of anti- Human Fc-Cy5 secondary antibody was subjected to secondary detection. This FMAT screen was identified via a 2200 anti-ST2 specific binding agent from a hybridoma produced by immunizing mice with the extracellular domain of ST2.

Interferon gamma interleukin release assays were then used to further characterize the ability of the 3400 anti-ST2-specific hybridoma supernatants of this combination to functionally antagonize ST2 signaling. Briefly, purified human peripheral blood mononuclear cells (PBMNC) or purified human NK cells were seeded into 96-well tissue culture plates and stimulated with human IL-33 and IL-12 to induce release of interferon-gamma to the supernatant. In the liquid. The interferon gamma content in the supernatant was quantified and directly correlated with Il-33/ST2-dependent signaling. Using this bioassay, hybridoma samples were tested for their ability to block interferon gamma release via blocking the ST2 signaling pathway. This screen identified 578 hybridoma supernatants from the ST2-Fc immunosuppression that inhibited the release of interferon gamma by more than 80%. In addition, 505 hybridoma supernatants which were immunogenized from the ST2Fc/IL-33 complex and which inhibited the release of interferon gamma by 70% or more were identified.

The cross-reactive binding of 1083 hybridoma supernatants of this group to mouse and cynomolgus ST2 was then further characterized for relative affinity fractionation, characterized by restriction antigen ELISA for biochemical receptors/ligands. Blocking, characterization by ELISA, and characterization by endogenous binding by FAC using cell lines. Using the data generated in these secondary analyses, the large group was diversified into two groups consisting of 40 hybridoma lines, which were further sub-selected, amplified and purified.

Example 3: K _D Determination

In this example, the affinity of the ST2 binding antibody is determined. Surface plasmon resonance evaluation was performed using a Proteon XPR-36 optical biosensor equipped with a GLC sensor wafer (Bio-Rad). Biosensor analysis was performed in a HBS-EP+ (1x) buffer system (10 mM HEPES pH 7.4, 150 mM NaCl, 3.0 mM EDTA, 0.05% Surfactant P20, GE Heathcare) at 25 °C. All reagents were maintained at 8 °C prior to injection.

Goat anti-human IgG via standard amine coupling (with Fc fragment specificity, Jackson ImmunoResearch) 1-6 lanes (about 4000 RU) fixed to the sensor surface in the vertical direction and subsequently blocked with ethanolamine. The antibody is captured (about 40-100 RU) to 1-5 lanes in the vertical direction. Vertical track 6 is left blank and used for reference purposes. Data were collected for groups of 15 antibodies (3 groups, 5 each).

The ST2 reagent (human or cynomolgus monkey) was prepared in running buffer to a concentration of 25 nM and then diluted 3 fold to 309 pM. A single injection in the horizontal direction delivers a full concentration series of ST2 molecules, using a buffer to complete a list of 6 samples and providing an in-line blank of double reference reaction data. Association (3 min) and dissociation (30 min) rates were monitored at a flow rate of 100 uL/min.

The surface was regenerated with 10 mM glycine (pH 1.5, 30 uL) at a flow rate of 100 uL/min.

Baseline correction, cropping, alignment, and interspot subtraction were performed on the data, and then fitted to a 1:1 binding model using ProteOn Manager (version 2.1.0.05). The results are shown in Table 4.

The affinity of other antibodies was determined using a slightly modified plasma resonance protocol. Surface plasmon resonance assessments were performed on Ab1, Ab2, Ab3, and Ab4 using a Biacore 3000 instrument (Biacore International AB, Uppsala, Sweden) equipped with a CM5 sensor wafer at 25 °C. Co-immobilization of anti-Fcγ-specific capture antibodies onto CM4 wafers using standard amine coupling chemistry using HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20, GE Heathcare) as running buffer Two flow cells. Briefly, each flow cell was activated with a 1:1 (v/v) mixture of 0.1 M NHS and 0.4 M EDC. AffiniPure goat anti-human IgG Fc gamma fragment specific antibody (Jackson ImmunoResearch, West) Grove, PA) was fixed to two flow cells at 30 ug/ml in 10 mM sodium acetate (pH 5.0) with a target amount of 3,000 RU. The residual reactive surface was deactivated by injection of 1 M ethanolamine. For all remaining steps, The running buffer was then converted to HBS-EP + 0.1 mg/ml BSA.

Triplicate triplicate of all antibodies were prepared in running buffer and diluted 3 fold and injected so that a 3 minute injection at 10 μl/min on the test flow cell resulted in approximately 75-90 reaction unit antibody capture in the test flow cell On the surface. No antibodies were captured on the surface of the control flow cell. Different concentrations (200 nM - 0.0914 nM) of human or cynomolgus ST2 were then flowed onto the two flow cells along with the buffer blank. A flow rate of 50 ul/min and a 2 minute association phase followed by a 4 minute dissociation phase were used. Injection 50uL 10 after each cycle mM glycine (pH 1.5) regenerates the surface. Fresh antibodies are then captured on the test flow cell to prepare for the next cycle. Separate long dissociation experiments (60 min) were performed in triplicate at a concentration of 200 nM.

The data was double-referenced by subtracting the control surface response to remove the bulk refractive index change and then subtracting the average buffer blank reaction to remove systemic artifacts from the experimental flow cell. Data was processed in BIA evaluation software v 4.1ST2 (Biacore International AB, Uppsala, Sweden) using local Rmax and fitted globally to a 1:1 interaction model. The association (k _a ) and dissociation (k _d ) rate constants were determined and used to calculate the dissociation equilibrium constant (K _D ). The dissociation rate constants and dissociation equilibrium constants of Ab1, Ab2, Ab3 and Ab4 are summarized in Table 5.

Example 4: pH sensitive binding of antibodies

A therapeutic antibody that binds to a target with reduced affinity at low pH may have enhanced PK properties which would allow it to be delivered less frequently or at lower doses. ( Nat Biotechnol. 2010 28(11): 1203-7 T.Igawa et al. Anti-reactive recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization), because lysosomes cause antibody release at low pH , followed by subsequent target degradation and antibody recycling.

Biosensor analysis of pH-sensitive binding of antibodies Ab1, Ab2, Ab3 and Ab4 was performed on Biacore 4000. Example 3 is similar to the set, wherein the K _D measured embodiments of these antibodies, only the data for a single concentration of antibody αST2 the same injection twice 2.46nM fit. The association (5 min) rate at pH 7.4 and the dissociation (10 min) rate at pH 5.5 and 7.4 were monitored at a flow rate of 30 uL/min. The reference subtraction data is fitted to the 1:1 model in Scrubber. Several antibodies exhibited significantly faster dissociation rates at lower pH, as shown in Table 6.

Example 5: Antibody cross-competition

A common way to characterize epitopes is through competition experiments. Antibodies that compete with each other can be considered to bind to the same site on the target. This example illustrates the method of determining competition for binding to ST2 and the results of the method as applied to the various antibodies described herein.

Binning experiments can be performed in a number of ways, and the methods used can have an effect on the results of the analysis. Common to these methods is that ST2 is typically bound by one reference antibody and probed with another antibody. If the reference antibody blocks the binding of the probe antibodies, the antibodies are considered to be in the same bin. The order in which antibodies are used is very important. If antibody A is used as a reference antibody and it blocks the binding of antibody B, the opposite is not always the case. Li: Antibody B is not necessarily blocked by antibody B. There are a number of factors at work: binding of antibodies can result in conformational changes in the target, which prevents binding of the second antibody, or epitopes that overlap each other but are not fully occluded, may result in a sufficiently high affinity for the second antibody and the target. Sexual interactions allow for binding. In general, antibodies can be considered to be binned if competition is observed in either order, and if the two antibodies are blocked from each other, the epitopes may overlap more completely.

For this example, a modified version of the multiple binning method described by Jia et al. (J. Immunological Methods, 288 (2004) 91-98) was used. Use soluble ST2-FLAG His. Streptavidin-coated Luminex beads (Luminex, number L100-L1XX-01, XX designated bead code) at 100 ul 6 pg/bead for each bead code at room temperature and in the dark The biotinylated monovalent mouse anti-human IgG capture antibody (BD Pharmingen, accession number 555785) was incubated for 1 hour and then washed 3x with PBSA (phosphate buffered saline (PBS) + 1% bovine serum albumin (BSA)). The beads of each code were individually incubated with 100 ul of an anti-ST2 antibody (coated antibody) diluted 1:10 for 1 hour and then washed. The beads were pooled and then dispensed into a 96-well filter plate (Millipore, number MSBVN1250). 100 ul 2 ug/ml ST2 was added to half of the well and buffer was added to the other half of the well and incubated for 1 hour before washing. 100 ul of an anti-ST2 antibody (detection Ab) diluted 1:10 was added to a well containing ST2 and a well without ST2, incubated for 1 hour, and then washed. Unrelated human IgG (Jackson, 009-000-003) and no antibody conditions (blank) were run as negative controls. 20 ul of PE-conjugated monovalent mouse anti-human IgG (BD Pharmingen, accession number 555787) was added to each well and incubated for 1 hour, followed by washing. The beads were resuspended in 75 ul PBSA and at least 100 event/bead codes were collected on a BioPlex instrument (BioRad).

The median fluorescence intensity (MFI) of the corresponding antibody pair without ST2 was subtracted from the signal containing the ST2 response. For antibody pairs that are considered to be simultaneously bound and therefore present in different bins, the response values must meet the following two criteria: 1) In either case, the value must be 2 times the coated antibody, irrelevant antibody or blank paired with itself, and 2) the value must be greater than the signal of the detection antibody coexisting with the unrelated antibody or blank coated beads. A minimum of three boxes were found, as shown in Table 7 below.

Example 6: IL-33 blocking analysis

Two AlphaScreens were used to explore the mechanism of action of the ST2 antibody. These assays are used in combination to determine whether an antibody inhibits the association of IL-33 with ST2, or conversely, whether the antibody specifically blocks the association of the co-receptor ST2 with AcP while still allowing IL-33 to associate with ST2 Hehe. AlphaScreen is an acronym for the screening of Amplified Luminescent Proximity Homogenous Assay.

In the first screening, the ability of the antibody to block the association between IL-33 and ST2 was assessed. This assay measures anti-ST2 antibody blocking biotinylated human IL-33 (coupled with streptavidin donor beads) and human ST2 with 6 x histidine tag (with Ni-chelate acceptor beads) Granular coupling) the ability to associate. IL-33/ST2 AlphaScreen was performed using a 40 ul reaction in a 96-well half-plate (Perkin Elmer). The assay buffers used for both AlphaScreens contained 40 mM HEPES (pH = 7.4), 1 mM CaCl2, 0.1% BSA, 0.05% Tween-20, and 100 mM NaCl. Each assay well contained 0.3 nM biotinylated human IL-33, 0.3 nM human ST2-FH (FH for FLAG and 6x histidine label), 10 ug/ml streptavidin coated donor beads (Perkin Elmer, Waltham, MA), 10 ug/ml Ni-chelate coated acceptor beads (Perkin Elmer) and 12.5 ug/ml anti-ST2 Ab. After all assay components were added, the plates were incubated overnight at room temperature in the dark. The next day, the plates were read on a 2103 Envision multi-label reader (Perkin Elmer). Laser excitation at 680 nm using donor beads to generate active oxygen that initiates a luminescence/fluorescence cascade in the acceptor beads that are closely adjacent due to the interaction of the bead-coupled proteins Thereby causing the emission of light detected at 570 nm.

In the second analysis, the ability of the antibody to inhibit the association of IL-33 mediated ST2 with the co-receptor AcP was assessed. This assay measures anti-ST2 antibody blocking IL-33-mediated biotinylated human ST2-Fc (coupling with streptavidin donor beads) and human AcP with 6 x histidine tag (with Ni - Chelate acceptor bead coupling) ability to associate. ST2/AcP was performed in 8 ul of reaction in 384-well opaque microporous (optiplate, Perkin Elmer) AlphaScreen. Each assay well contained 5 nM human IL-33, 5 nM biotinylated human ST2-Fc, 5 nM human AcP-FH, 10 ug/ml streptavidin coated donor beads, 10 ug/ml Ni-chelate Coated acceptor beads and 12.5 ug/ml anti-ST2 Ab. After all assay components were added, the plates were incubated overnight at room temperature in the dark. The next day, the plates were read on a 2103 Envision multi-label reader (Perkin Elmer) using the same parameters as the first analysis above.

The results of the two AlphaScreens are presented in Table 8 below. Inhibition of each antibody was presented as a percentage of inhibition of the signal in the AlphaScreen of the given antibody using a concentration of 12.5 ug/ml relative to the signal in the assay when the antibody was not included in the assay well. Some antibodies more completely inhibit the interaction of ST2 and IL-33 than their inhibition of ST2/IL-33/AcP interaction, and some antibodies inhibit ST2/ more completely than inhibiting the interaction between ST2 and IL-33. IL-33/AcP interaction. All antibodies inhibited the interaction of IL-33 with ST2 by at least 50%.

Example 7: Biological analysis of human IL-33 in vitro

Exemplary ST2 human mAbs were tested in human bioassays using purified CD4+ T cells obtained from various donors stimulated with human IL-33 and human IL-2. The analysis procedure is as follows. The cells were seeded at a volume of 60 ul at 250,000 cells/well in a 96-well round bottom plate. After pre-incubation, 30 ul of 4 x huIL-2+huIL-33 mixture was added to each well. The total volume in the 96-well round bottom plate is 120 ul. The antibody was started at 20 ug/ml and diluted 1:3 to generate a 10-point curve. Achieved 4 ×, a total of 30ul. After pre-incubation of the Ab and cells, 30 ul of 4 x huIL-2+ huIL-33 mixture was added to each well. 37 ° C, 5% CO 2 for 48 hours. Harvest the supernatant. Inhibition of IL-5 was analyzed by huIL-5 ELISA.

Figure 3 shows inhibition of IL-5 production induced by human IL-33 by ST2 mAbs from various donor CD4+ T cells. The (-) line depicts positive control values for the combination of human IL-33 and human IL-2 without inhibition. (....) shows the positive control value of human IL-2. The (--) line shows the medium control value. Human CD4+ T cells were pre-incubated with anti-ST2 mAb for 30 minutes and then stimulated with human IL-33 (4 ng/ml) and human IL-2 (10 ng/ml) for 48 hours. Figure 3 shows that ST2 antibodies are capable of inhibiting ST2 activation induced by human IL-33, as determined by IL-5 production from CD4+ T cells. The ST2 antibody is capable of antagonizing IL-33-induced IL-5 production by CD4+ T cells with an IC50 of about <100 nM. Table 9 shows representative IC50 values.

Example 8: Analysis of IFNγ release in cynomolgus CD4+ T cells

Peripheral blood mononuclear cells (PBMC) of cynomolgus monkeys were enriched by normal donor peripheral blood treated with acidic citrate dextrose (ACD) by gradient centrifugation at ISOLYMPH (Gallard-Schlesinger Industries, Plainview, NY). Subsequent isolation of cynomolgus CD4+ T cells was performed using the Miltenyi Biotec cynomolgus CD4+ T cell isolation kit. Isolated cynomolgus CD4+ T cells (2 x 10 ⁵ cells/well in 96-well plates) were incubated with different concentrations of purified monoclonal antibodies for 30 minutes at room temperature and subsequently with IL-33 (10 ng) /mL), IL-2 (10 ng/mL) and IL-12p70 (50 ng/mL) were stimulated for 84 hours. The presence of cynomolgus IFNγ in the resulting cell-free cynomolgus CD4+ T cell culture supernatant was then analyzed by ELISA (example data is provided in Table 10). The efficacy of purified monoclonal antibodies was determined from 3 individual donors in the cynomolgus CD4+ T cell IFNy release assay.

Example 9: Analysis of IL-8 release from human eosinophils

Human red blood cells and granules were enriched from heparinized normal donor peripheral blood by gradient centrifugation with ISOLYMPH (Gallard-Schlesinger Industries, Plainview, NY). Red blood cells were removed using ACK lysis buffer (Gibco, Carlsbad, CA). Subsequent isolation of eosinophilic leukocytes was performed using the eosinophilic leukocyte separation kit of Miltenyi Biotec. Separation of eosinophilic leukocytes (2 x 10 ⁵ cells/well in 96-well plates) at room temperature with several dilutions of non-pure or pure supernatant or different concentrations of purified monoclonal antibodies Incubate for 30 minutes and then stimulate with IL-33 (2 ng/mL) and IL-3 (100 ng/mL) for 3 days. The presence of IL-8 in the resulting cell-free eosinophil culture supernatant was then analyzed by ELISA. The example data is shown in Table 11. The efficacy of purified monoclonal antibodies was determined from 3 individual donors in the eosinophilic IL-8 release assay.

Example 10: Efficacy of anti-ST2 antibodies compared to commercially available antibodies Dose response of human IL-33 in human NK cell analysis.

With human IL-12 (1ng / mL) + increased amount of human IL-33 treated primary human CD56-positive NK cells (5 × 10 ⁴ cells), as shown in FIG. 4. After 22 hours, cell free supernatants were collected and IFN-[gamma] concentrations were measured using commercial analysis (R&D Systems). 10 ng/mL IL-33 was used as a stimulating dose for ST2 antibody inhibition.

Inhibition of IL-33 activity by antibodies

Stimulate human NK cells as above. The ST2 antibody was added to the cells at a concentration as shown in Figure 5 30 minutes before the addition of IL-33 and IL-12. Twenty-two hours after IL-33 treatment, cell-free supernatants were collected and IFN-γ concentrations were measured using commercial analysis (R&D Systems). Indicates the pure name of a commercially available antibody. Only Ab2 completely inhibited the IL-33 dependent IFN-γ response and it was significantly more potent than any of the commercially available huST2 antibodies. The IC50 values corresponding to each antibody are shown in Table 12.

Example 11: ST2 alanine/arginine scanning mutagenesis

This example characterizes ST2 antibodies based on the effect of ST2 mutagenesis on the ability of ST2 antibodies to bind to targets. As previously indicated by the binding data, ST2 domains 1 and 2 are primarily responsible for antibody binding of the antibody panel analyzed by ST2 scanning mutagenesis in this example. Therefore, only the domains 1 and 2 (D1D2) of ST2 are structurally considered in the context of the full-length ST2 in the design of the mutation site.

The ST2 and IL-33 composite model coordinates were obtained from Lingel et al., Structure (London, England: 1993). Elsevier Ltd 17 , 1398-410. Calculating solvent accessibility per side chain of ST2 in a molecular operating environment ( Molecular Operating Environment (MOE), 2011.10; Chemical Computing Group , 1010 Sherbooke St. West, Suite 910 , Montreal, QC, Canada, H3A 2R7, 2011 ). And then the use of such solvent by first selecting all values having the least exposed D1D2 residue or a side chain having at least 10Å ² 10Å ² total exposure of glycine residues selected for mutagenesis D1D2 of surface residues. The removal of glycine residues with a positive π angle is equally applicable to the proline residues, so that mutations at equal positions have a higher probability of distorting the local protein structure. The cysteine residue is also selected to remove the disulfide linkage. Residue A82 was removed after visual inspection. This method produces a list of 140 residues for mutagenesis. All residues were mutated to arginine, which was mutated to alanine except for arginine and lysine residues.

Expression of all mutants of the His-Avi-tagged parental ST2 extracellular domain (ECD) in the pTT5 vector in transiently transfected 293-6E suspension cells (NRCC) in 24-well plates Building. In vivo biotinylation was achieved by co-transfection of BiR A in the pTT5 vector. The supernatant was dialyzed against PBS to remove excess biotin.

BioPlex binding assays were used to measure the binding of anti-ST2 antibodies to point mutation ST2. The biotinylated mutant was bound to the 80-bead code of streptavidin coated beads (Luminex, number L100-L1XX-01, XX designated bead code). The 80 bead code allows for multiplexing of two groups of 70 mutants (140 mutants in total). Each group included 6 parental controls, 3 unrelated protein controls, and 1 blank. The antibody binding of the mutant protein binds to the antibody of the parent protein.

100 ul of ST2 mutant diluted with 1:7 and pre-bound beads, parental and control or protein-free wash 5×, pooled and aliquoted into 96-well filter plates (Millipore) with PBS + 1% BSA, then again washing. 100 ul of the 3-fold diluted anti-ST2 antibody was added to 3 identical wells, incubated for 1 hour at RT and washed. 100 ul of PE-conjugated anti-human IgG Fc (Jackson, number 109-116-170) diluted 1:500 was added to each well, incubated for 0.5 hours and washed. The beads were resuspended in 75 uL of solution, shaken for at least 3 min, and read on BioPlex.

A validation experiment was performed to evaluate the "bead area" - "bead area" (B-B) variability prior to running the binding analysis. In the validation experiments, all beads were conjugated to the same wild type control protein. Therefore, the difference between the bead regions is simply due to the B-B variance and is not confused with the difference between the wild-type protein and the mutant protein. The titration of the antibody was run with 12 replicates in different wells.

The goal of this statistical analysis is to estimate the B-B variability of the estimated EC50 for the binding curve. The estimated B-B standard deviation (SD) was then used to construct the EC50 confidence interval for wild-type and mutant proteins during the curve comparison experiment.

A four parameter logistic model was fitted to the binding data for each bead region. Use the resulting curve quality control (QC) results and the top (max), bottom (min), hill slope (slope) and EC50 (xmid) natural logarithm of the parameter estimates The "sumout.xls" file is used as the raw data for analysis. The B-B variability of each parameter was then estimated by fitting the mixed effect model using the SAS PROC MIXED program. Only curves with a "good" QC state are included in the analysis. The final mixed-effects model includes only the residual portion (ie, the individual bead regions) as a random effect. The least square mean (LS-means) of each parameter is also estimated by a mixed effect model. The B-B SD is calculated by taking the square root of the B-B variance. The fold change between LS-mean + 2SD and LS-mean-2SD (which represents approximately the upper side of the population and the 97.5 percentile of the lower side) was also calculated.

For each antibody, to identify mutants that did not produce a large amount of response relative to the WT control, mutants with max(MFI) less than 30% WT control max (MFI) were identified and marked as hit max.

The EC50 of the mutant binding curve was compared to the wild type binding curve. For further consideration, statistically significant differences were identified as hits. Curves with the "no fit" or "bad fit" signs are excluded from this analysis.

Two sources of change were considered in the comparison of EC50 estimates, namely curve fit changes and inter-bead changes. Wild-type and mutant systems are linked to different beads, so the difference is confused with the difference between the beads. The curve fit change is estimated by the standard error of the log EC50 estimate. Inter-bead changes were determined experimentally using experiments that attached wild-type controls to each bead. The changes in the beads of the EC50 estimates of the wild-type binding curves of this experiment were used to estimate the inter-bead changes in the actual positioning experiments.

A comparison of the two EC50s (on a logarithmic scale) was performed using the Student's t-test. The t statistic is calculated as the ratio between δ (the absolute difference between the EC50 estimates) and the standard deviation of δ. The variance of δ is estimated from the sum of the three components (the EC50 variance estimate for the mutant and wild-type curves in the nonlinear regression and the two-bead variance estimated from the individual experiments). The doubling of the inter-bead variance is due to the assumption that the mutant has the same variance as both wild-type beads.

Using the Satterthwaite’s approximation (1946) Calculate the degree of freedom of the standard deviation of δ.

Individual p-values and confidence intervals (95% and 99%) were obtained from the Stuart's t-distribution of each comparison.

In the case of multiple wild-type controls, conservative treatments were performed by selecting wild-type controls that were most similar to the mutants (i.e., selecting the one with the greatest p-value).

When a large number of tests are performed simultaneously, multiplicity adjustment is very important for controlling false positives. Two forms of multiplicity adjustment are implemented for this analysis: Family Error (FWE) Control and False Discovery Rate (FDR) Control. The FEW process controls one or more possibilities that are not truly hits; the FDR process controls the expected proportion of false positives in the selected hits. The former treatment method is more conservative than the latter and is not as effective as the latter. There are a number of methods available for both treatments. For this analysis, the Hochberg's method (1988) was selected for FWE analysis and the Benjamini-Hochberg FDR method (1995) was selected for FDR analysis. The adjusted p values for each antibody or the entire assay in both treatments were calculated.

Mutants were selected to have an effect by the following criteria: 1) returning a bad fit or no fit result for the mutant, and 2) selecting a mutant for the hit max criterion. 3) Family error p value is less than 0.01 or 4) Bmax value is greater than 200% parent. If the effect decreases Bmax or increases the EC50 value, the hit is called an inhibitor, and if the effect increases Bmax or decreases the EC50 value, the hit is called an activator. Eight mutations were excluded from the hit list by the effect of >90% of the tested antibodies, which were: K37A, R46A, D63R, V71R, G106R, K112A, N132R, Q137R and Y141R.

The results of the analysis are provided in Tables 13 and 14.

Example 12: Hydrogen/deuterium exchange (HDX) analysis

In this example, Ab2 binds to ST2 and determines the effect of binding on HDX.

Transition in mammalian 293-6E cells with soluble ST2 protein (containing domain 1-3 of amino acid 19-322 of SEQ ID NO: 1) having both FLAG-tag and His-tag at the C-terminus Purified by IMAC (fixed metal ion affinity chromatography) and further purified by preparative SEC (size exclusion chromatography). The protein was then concentrated to 3.5 mg/mL using ultrafiltration. ST2 antibody Ab2 was expressed in engineered CHO-CS9 cells and purified by protein A affinity chromatography followed by preparative SEC. Analytical SEC was used to determine 0.75: 1.00 molar ratio of antibody: antigen is optimal for ensuring complete binding of the ST2 protein to the system. Both the free ST2 protein and the antigen-antibody complex were stored in PBS buffer (pH 7.2).

The HDX experiment was performed on an automated HDX system (Zhang, Zhang et al. 2012). Briefly, the H/D exchange process was diluted with 5 uL of free ST2 protein solution (3.5 mg/mL) or ST2-antibody complex (with a jST2 concentration of 3.5 mg/mL and an antigen:antibody ratio of 1:0.75). This was started in 25 uL of D ₂ O (pH 7.2) buffer in 100 mM PBS prepared by dissolving PBS tablets in D ₂ O water at 25 °C. For each HDX experiment, the exchange reactions were incubated for different label durations (30 sec, 2 min, 8 min, 30 min and 2 hr, 8 hr) and quenched/denatured by mixing 20 μL of labeling solution with 80 μL at 1 °C. /Reducing buffer (7.25 M urea and 625 mM hydrazine (2-carboxyethyl) phosphine (TCEP), 0.45 M glycine (pH 2.7)) to quench the labeling reaction.

A 40 [mu]L aliquot of the quenching solution was transferred to 120 [mu]L of 0.4 mg/mL porcine pepsin (Sigma, St. Louis, MO) solution. The digestion solution was immediately injected into the sample loop at 1 ° C for 6 min for complete digestion. In addition, the digest was separated by a C18 column (3 series columns, BEH C18, 2.1 mm x 5 mm, Waters, Milford, MA). HPLC separation was performed using a 5-min 1-40% ACN gradient at 1 °C. LC eluents were analyzed by Orbitrap Elite mass spectrometer (Thermo Scientific, San Jose, CA) in a data dependent LC-MS/MS experiment.

Deuterium labeling, quenching, proteolytic digestion and injection were performed on a LEAP HD-X PAL system controlled by LEAP Shell (LEAP Technologies, Carrboro, NC).

The experiment was repeated 3 times, and in each experiment, each time point was repeated twice. A standard peptide mixture was added to the sample to track and correct the return exchange variability. The mixture contains the three peptides: bradykinin, angiotensin I and leucine enkephalin. A specially designed tetrapeptide PPPI (synthesized by AnaSpec, Fremont, CA) was used as a second internal standard to minimize inter-run variability. The H2D-free ST2 protein digest was also analyzed as a control.

The resulting data file is processed using a mass analyzer program (Zhang, Zhang et al. 2012). The software identifies the peptide from the antigen digest, calculates the exchange rate of each peptide, corrects the exchange information obtained from the internal standard, and fits the data into the model to obtain the protection factor of each residue.

Comparison of the exchange profile between free ST2 and antibody-bound ST2 proteins reveals two regions of interest and can be potential epitopes. The two regions of the ST2 sequence are refined to IVRCPRQGKPSY (amino acid 33-44 of SEQ ID NO: 1, corresponding to amino acid 15-26 of mature ST2) and IVRSPTF (SEQ ID NO) from multiple overlapping peptides Amino acid 88-94 of 1 corresponds to amino acid 70-76 of mature ST2). When ST2 is in a free state, the regions are less protected and the exchange rate is significantly reduced after ST2 and he antibody binding. Furthermore, based on the ST2 homology structure model shown in Figure 6, the two sequence extensions occupy similar spatial positions on the outer surface of the protein. These results led to the conclusion that both peptides are involved in the binding between the Ab2 and ST2 proteins.

Example 13: X-ray crystallography

In this example, the crystal structure of the complex of the sc-dsFv fragment of Ab2 and ST2 provides a specific amino acid residue in the interaction interface.

Ab2 sc-dsFv was expressed in BL21 (DE3) star cells. Briefly, Ab2 sc-dsFv comprising connected by a connecting member (G ₄ S) ₃ to a heavy chain variable domain of the light chain variable domain. To further stabilize the molecule, the disulfide bond is engineered into the molecule by mutation of position 44 of the heavy chain variable region and position 101 of the light chain variable region to cysteine. This protein appears as an inclusion body. The inclusion bodies are dissolved and refolded. Proteins were further purified on size exclusion column (SEC) and ion exchange MonoQ columns and subsequently polished on SEC.

ST2 is expressed in 293S cells. Protein was purified by Ni-affinity column and deglycosylated using EndoH. The protein was further purified on SEC.

A complex of Ab2 sc-dsFv and ST2 was formed by mixing Ab2 sc-dsFv with excess ST2. The complex was purified on SEC and concentrated to 12 mg/ml for crystallization. The protein complex was crystallized in 32-36% PEG400 and 0.1 M Hepes (pH 7.5-8.5) using a drop-wise vapor diffusion method at 16 °C.

A 1.9 Å resolution data set was collected at Advanced Light Source, Lawrence Berkeley National Lab (Berkeley, CA). Data was processed using MOSFLM ( Leslie, 1992 ) and scaled by SCALA (Collaborative Computational Project, No. 4 (1994)) in the CCP4 suite. The structure was resolved by a molecular replacement method using the program PHASER ( Cuney et al., 2007 ) using the D1D2 domain of the published structure of IL-1RII (pdb code: 3O4O) and the variable domain of the Fab structure of Ab2 as a research model. Iterative structural refinement and model construction were performed using REFMAC5 (Murshudov et al., 1997) and COOT ( Esley and Cowtan, 2004 ).

Interface analysis was performed using the programs PISA, AREAMOL, and NCONTACT in the CCP4 package. The map was refined using Pymol (The PyMOL Molecular Graphics System. Schrödinger).

The crystal structure of the ST2/Ab2 sc-dsFv complex was resolved to a resolution of 1.9 Å. There are two separate ST2/Ab2 sc-dsFv complex pairs in the asymmetric unit (Figure 7). Each complex consists of one ST2 molecule and one Ab2 sc-dsFv fragment. The ST2 molecule consists of two IgG-like domains (D1 and D2 domains). The Ab2 Fv domain utilizes all six CDR loops of the heavy and light chains to interact with the ST2 molecule (Figure 8). For ST2, the two loops of ST2 (loop BC and loop FG) and the N-terminus interact directly with the antibody. The total hidden solvent accessible surface area between ST2 and Ab2 sc-dsFv is 1803 Å ² .

The interface between ST2 and Ab2 is highly charged. ST2 has a cluster of base residues (Lys and Arg) in the D1 domain. This positively charged surface patch is complementary to the negatively charged patch formed by the acidic residue clusters (Asp and Glu) in the CDR regions on Ab2 (Fig. 9).

Two different methods are used to define interface residues. In the first method, solvent exposure differences are used to define interfacial residues. The solvent accessible surface area of each residue of ST2 (or Ab2 sc-dsFv) in the complex is calculated and compared to the solvent accessible surface area of the corresponding residue in ST2 (or Ab2 sc-dsFv) stripped from the complex. Amino acids with all of the differences of ST2 and Ab2 greater than 10% are shown in Tables 15 and 16, respectively. The surface exposure difference of Arg72 and Tyr81 of ST2 is less than 10%. However, detection of the complex structure revealed that both residues form a water-mediated hydrogen bond with the Ab2 heavy chain residue and are therefore included in the list.

In the second method, an interface residue having at least one atom within a predetermined distance from its target protein is selected. Two shells are defined based on different distance cutoff values.

The core shell includes all residues up to 5.0 Å apart.

The boundary shell includes all residues that are longer than 5.0 Å but shorter than 8.0 Å.

A complete list of amino acid residues in the shells of the heavy and light chains of ST2 and Ab2 are shown in Tables 17, 18 and 19, respectively. For residues that form a hydrogen bond or a salt bridge with a subject protein, the type of specific interaction is represented as a residue post-bracket (HB represents a hydrogen bond and SB represents a salt bridge).

Epitope residue matching defined by solvent exposure differences is defined by distance cutoff The residue of the nuclear interaction group. Table 20 lists all hydrogen bonds and salt bridge pairs within the interface. Table 20 interactions in the interface

The ST2 epitope region obtained from the HDX-MS analysis of Example 12 was confirmed by crystallographic data. Two epitopes (15-26 and 70-76) from HDX were identified as epitopes in higher resolution crystallographic data. Specifically, Arg20, Gly22, Lys23 and Tyr26 and Thr75 were identified as being close to the antibody at a distance of less than 3.4 Å. Other residues (Pro 19, Gln21, Ile70, Val71, Arg72, Ser73 Pro74, and Phe76) identified as being between 3.4 Å and 5 Å from the antibody are also encompassed in the HDX epitope.

In summary, the results confirmed that the ST2 epitopes obtained from both HDX-MS and crystallography were consistent. The BC loop and the FG loop (see crystallographic data) in domain 1 are the major interaction sites.

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<211> 1329

<212> DNA

<213> Homo sapiens

<400> 10

<210> 11

<211> 1341

<212> DNA

<213> Homo sapiens

<400> 11

<210> 12

<211> 1344

<212> DNA

<213> Homo sapiens

<400> 12

<210> 13

<211> 1344

<212> DNA

<213> Homo sapiens

<400> 13

<210> 14

<211> 1341

<212> DNA

<213> Homo sapiens

<400> 14

<210> 15

<211> 1326

<212> DNA

<213> Homo sapiens

<400> 15

<210> 16

<211> 1326

<212> DNA

<213> Homo sapiens

<400> 16

<210> 17

<211> 1326

<212> DNA

<213> Homo sapiens

<400> 17

<210> 18

<211> 444

<212> PRT

<213> Homo sapiens

<400> 18

<210> 19

<211> 447

<212> PRT

<213> Homo sapiens

<400> 19

<210> 20

<211> 448

<212> PRT

<213> Homo sapiens

<400> 20

<210> 21

<211> 443

<212> PRT

<213> Homo sapiens

<400> 21

<210> 22

<211> 447

<212> PRT

<213> Homo sapiens

<400> 22

<210> 23

<211> 448

<212> PRT

<213> Homo sapiens

<400> 23

<210> 24

<211> 448

<212> PRT

<213> Homo sapiens

<400> 24

<210> 25

<211> 447

<212> PRT

<213> Homo sapiens

<400> 25

<210> 26

<211> 442

<212> PRT

<213> Homo sapiens

<400> 26

<210> 27

<211> 442

<212> PRT

<213> Homo sapiens

<400> 27

<210> 28

<211> 442

<212> PRT

<213> Homo sapiens

<400> 28

<210> 29

<211> 118

<212> PRT

<213> Homo sapiens

<400> 29

<210> 30

<211> 121

<212> PRT

<213> Homo sapiens

<400> 30

<210> 31

<211> 122

<212> PRT

<213> Homo sapiens

<400> 31

<210> 32

<211> 117

<212> PRT

<213> Homo sapiens

<400> 32

<210> 33

<211> 120

<212> PRT

<213> Homo sapiens

<400> 33

<210> 34

<211> 122

<212> PRT

<213> Homo sapiens

<400> 34

<210> 35

<211> 122

<212> PRT

<213> Homo sapiens

<400> 35

<210> 36

<211> 121

<212> PRT

<213> Homo sapiens

<400> 36

<210> 37

<211> 116

<212> PRT

<213> Homo sapiens

<400> 37

<210> 38

<211> 116

<212> PRT

<213> Homo sapiens

<400> 38

<210> 39

<211> 116

<212> PRT

<213> Homo sapiens

<400> 39

<210> 40

<211> 5

<212> PRT

<213> Homo sapiens

<400> 40

<210> 41

<211> 5

<212> PRT

<213> Homo sapiens

<400> 41

<210> 42

<211> 5

<212> PRT

<213> Homo sapiens

<400> 42

<210> 43

<211> 5

<212> PRT

<213> Homo sapiens

<400> 43

<210> 44

<211> 5

<212> PRT

<213> Homo sapiens

<400> 44

<210> 45

<211> 5

<212> PRT

<213> Homo sapiens

<400> 45

<210> 46

<211> 5

<212> PRT

<213> Homo sapiens

<400> 46

<210> 47

<211> 5

<212> PRT

<213> Homo sapiens

<400> 47

<210> 48

<211> 5

<212> PRT

<213> Homo sapiens

<400> 48

<210> 49

<211> 5

<212> PRT

<213> Homo sapiens

<400> 49

<210> 50

<211> 5

<212> PRT

<213> Homo sapiens

<400> 50

<210> 51

<211> 17

<212> PRT

<213> Homo sapiens

<400> 51

<210> 52

<211> 17

<212> PRT

<213> Homo sapiens

<400> 52

<210> 53

<211> 16

<212> PRT

<213> Homo sapiens

<400> 53

<210> 54

<211> 17

<212> PRT

<213> Homo sapiens

<400> 54

<210> 55

<211> 16

<212> PRT

<213> Homo sapiens

<400> 55

<210> 56

<211> 16

<212> PRT

<213> Homo sapiens

<400> 56

<210> 57

<211> 16

<212> PRT

<213> Homo sapiens

<400> 57

<210> 58

<211> 16

<212> PRT

<213> Homo sapiens

<400> 58

<210> 59

<211> 17

<212> PRT

<213> Homo sapiens

<400> 59

<210> 60

<211> 17

<212> PRT

<213> Homo sapiens

<400> 60

<210> 61

<211> 17

<212> PRT

<213> Homo sapiens

<400> 61

<210> 62

<211> 9

<212> PRT

<213> Homo sapiens

<400> 62

<210> 63

<211> 12

<212> PRT

<213> Homo sapiens

<400> 63

<210> 64

<211> 14

<212> PRT

<213> Homo sapiens

<400> 64

<210> 65

<211> 8

<212> PRT

<213> Homo sapiens

<400> 65

<210> 66

<211> 13

<212> PRT

<213> Homo sapiens

<400> 66

<210> 67

<211> 14

<212> PRT

<213> Homo sapiens

<400> 67

<210> 68

<211> 14

<212> PRT

<213> Homo sapiens

<400> 68

<210> 69

<211> 13

<212> PRT

<213> Homo sapiens

<400> 69

<210> 70

<211> 7

<212> PRT

<213> Homo sapiens

<400> 70

<210> 71

<211> 7

<212> PRT

<213> Homo sapiens

<400> 71

<210> 72

<211> 7

<212> PRT

<213> Homo sapiens

<400> 72

<210> 73

<211> 660

<212> DNA

<213> Homo sapiens

<400> 73

<210> 74

<211> 642

<212> DNA

<213> Homo sapiens

<400> 74

<210> 75

<211> 654

<212> DNA

<213> Homo sapiens

<400> 75

<210> 76

<211> 657

<212> DNA

<213> Homo sapiens

<400> 76

<210> 77

<211> 654

<212> DNA

<213> Homo sapiens

<400> 77

<210> 78

<211> 654

<212> DNA

<213> Homo sapiens

<400> 78

<210> 79

<211> 654

<212> DNA

<213> Homo sapiens

<400> 79

<210> 80

<211> 654

<212> DNA

<213> Homo sapiens

<400> 80

<210> 81

<211> 645

<212> DNA

<213> Homo sapiens

<400> 81

<210> 82

<211> 645

<212> DNA

<213> Homo sapiens

<400> 82

<210> 83

<211> 645

<212> DNA

<213> Homo sapiens

<400> 83

<210> 84

<211> 220

<212> PRT

<213> Homo sapiens

<400> 84

<210> 85

<211> 214

<212> PRT

<213> Homo sapiens

<400> 85

<210> 86

<211> 218

<212> PRT

<213> Homo sapiens

<400> 86

<210> 87

<211> 219

<212> PRT

<213> Homo sapiens

<400> 87

<210> 88

<211> 218

<212> PRT

<213> Homo sapiens

<400> 88

<210> 89

<211> 218

<212> PRT

<213> Homo sapiens

<400> 89

<210> 90

<211> 218

<212> PRT

<213> Homo sapiens

<400> 90

<210> 91

<211> 218

<212> PRT

<213> Homo sapiens

<400> 91

<210> 92

<211> 215

<212> PRT

<213> Homo sapiens

<400> 92

<210> 93

<211> 215

<212> PRT

<213> Homo sapiens

<400> 93

<210> 94

<211> 215

<212> PRT

<213> Homo sapiens

<400> 94

<210> 95

<211> 114

<212> PRT

<213> Homo sapiens

<400> 95

<210> 96

<211> 108

<212> PRT

<213> Homo sapiens

<400> 96

<210> 97

<211> 112

<212> PRT

<213> Homo sapiens

<400> 97

<210> 98

<211> 113

<212> PRT

<213> Homo sapiens

<400> 98

<210> 99

<211> 112

<212> PRT

<213> Homo sapiens

<400> 99

<210> 100

<211> 112

<212> PRT

<213> Homo sapiens

<400> 100

<210> 101

<211> 112

<212> PRT

<213> Homo sapiens

<400> 101

<210> 102

<211> 112

<212> PRT

<213> Homo sapiens

<400> 102

<210> 103

<211> 109

<212> PRT

<213> Homo sapiens

<400> 103

<210> 104

<211> 109

<212> PRT

<213> Homo sapiens

<400> 104

<210> 105

<211> 109

<212> PRT

<213> Homo sapiens

<400> 105

<210> 106

<211> 17

<212> PRT

<213> Homo sapiens

<400> 106

<210> 107

<211> 11

<212> PRT

<213> Homo sapiens

<400> 107

<210> 108

<211> 16

<212> PRT

<213> Homo sapiens

<400> 108

<210> 109

<211> 16

<212> PRT

<213> Homo sapiens

<400> 109

<210> 110

<211> 16

<212> PRT

<213> Homo sapiens

<400> 110

<210> 111

<211> 16

<212> PRT

<213> Homo sapiens

<400> 111

<210> 112

<211> 16

<212> PRT

<213> Homo sapiens

<400> 112

<210> 113

<211> 16

<212> PRT

<213> Homo sapiens

<400> 113

<210> 114

<211> 12

<212> PRT

<213> Homo sapiens

<400> 114

<210> 115

<211> 12

<212> PRT

<213> Homo sapiens

<400> 115

<210> 116

<211> 12

<212> PRT

<213> Homo sapiens

<400> 116

<210> 117

<211> 7

<212> PRT

<213> Homo sapiens

<400> 117

<210> 118

<211> 7

<212> PRT

<213> Homo sapiens

<400> 118

<210> 119

<211> 7

<212> PRT

<213> Homo sapiens

<400> 119

<210> 120

<211> 7

<212> PRT

<213> Homo sapiens

<400> 120

<210> 121

<211> 7

<212> PRT

<213> Homo sapiens

<400> 121

<210> 122

<211> 7

<212> PRT

<213> Homo sapiens

<400> 122

<210> 123

<211> 7

<212> PRT

<213> Homo sapiens

<400> 123

<210> 124

<211> 7

<212> PRT

<213> Homo sapiens

<400> 124

<210> 125

<211> 7

<212> PRT

<213> Homo sapiens

<400> 125

<210> 126

<211> 7

<212> PRT

<213> Homo sapiens

<400> 126

<210> 127

<211> 7

<212> PRT

<213> Homo sapiens

<400> 127

<210> 128

<213> Homo sapiens

<400> 128

<210> 129

<211> 9

<212> PRT

<213> Homo sapiens

<400> 129

<210> 130

<211> 8

<212> PRT

<213> Homo sapiens

<400> 130

<210> 131

<211> 9

<212> PRT

<213> Homo sapiens

<400> 131

<210> 132

<211> 8

<212> PRT

<213> Homo sapiens

<400> 132

<210> 133

<211> 8

<212> PRT

<213> Homo sapiens

<400> 133

<210> 134

<211> 8

<212> PRT

<213> Homo sapiens

<400> 134

<210> 135

<211> 8

<212> PRT

<213> Homo sapiens

<400> 135

<210> 136

<211> 9

<212> PRT

<213> Homo sapiens

<400> 136

<210> 137

<211> 9

<212> PRT

<213> Homo sapiens

<400> 137

<210> 138

<211> 9

<212> PRT

<213> Homo sapiens

<400> 138

<210> 139

<211> 1335

<212> DNA

<213> Homo sapiens

<400> 139

<210> 140

<211> 1347

<212> DNA

<213> Homo sapiens

<400> 140

<210> 141

<211> 1347

<212> DNA

<213> Homo sapiens

<400> 141

<210> 142

<211> 445

<212> PRT

<213> Homo sapiens

<400> 142

<210> 143

<211> 449

<212> PRT

<213> Homo sapiens

<400> 143

<210> 144

<211> 449

<212> PRT

<213> Homo sapiens

<400> 144

<210> 145

<211> 119

<212> PRT

<213> Homo sapiens

<400> 145

<210> 146

<211> 123

<212> PRT

<213> Homo sapiens

<400> 146

<210> 147

<211> 123

<212> PRT

<213> Homo sapiens

<400> 147

<210> 148

<211> 5

<212> PRT

<213> Homo sapiens

<400> 148

<210> 149

<211> 5

<212> PRT

<213> Homo sapiens

<400> 149

<210> 150

<211> 5

<212> PRT

<213> Homo sapiens

<400> 150

<210> 151

<211> 17

<212> PRT

<213> Homo sapiens

<400> 151

<210> 152

<211> 16

<212> PRT

<213> Homo sapiens

<400> 152

<210> 153

<211> 16

<212> PRT

<213> Homo sapiens

<400> 153

<210> 154

<211> 10

<212> PRT

<213> Homo sapiens

<400> 154

<210> 155

<211> 15

<212> PRT

<213> Homo sapiens

<400> 155

<210> 156

<211> 15

<212> PRT

<213> Homo sapiens

<400> 156

<210> 157

<211> 642

<212> DNA

<213> Homo sapiens

<400> 157

<210> 158

<211> 654

<212> DNA

<213> Homo sapiens

<400> 158

<210> 159

<211> 657

<212> DNA

<213> Homo sapiens

<400> 159

<210> 160

<211> 214

<212> PRT

<213> Homo sapiens

<400> 160

<210> 161

<211> 218

<212> PRT

<213> Homo sapiens

<400> 161

<210> 162

<211> 219

<212> PRT

<213> Homo sapiens

<400> 162

<210> 163

<211> 108

<212> PRT

<213> Homo sapiens

<400> 163

<210> 164

<211> 108

<212> PRT

<213> Homo sapiens

<400> 164

<210> 165

<211> 113

<212> PRT

<213> Homo sapiens

<400> 165

<210> 166

<211> 11

<212> PRT

<213> Homo sapiens

<400> 166

<210> 167

<211> 16

<212> PRT

<213> Homo sapiens

<400> 167

<210> 168

<211> 16

<212> PRT

<213> Homo sapiens

<400> 168

<210> 169

<211> 7

<212> PRT

<213> Homo sapiens

<400> 169

<210> 170

<211> 7

<212> PRT

<213> Homo sapiens

<400> 170

<210> 171

<211> 7

<212> PRT

<213> Homo sapiens

<400> 171

<210> 172

<211> 9

<212> PRT

<213> Homo sapiens

<400> 172

<210> 173

<211> 8

<212> PRT

<213> Homo sapiens

<400> 173

<210> 174

<211> 8

<212> PRT

<213> Homo sapiens

<400> 174

<210> 175

<211> 187

<212> PRT

<213> Homo sapiens

<400> 175

Claims (62)

An isolated ST2 antibody comprising: a) a light chain variable domain having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 96; b) a heavy chain variable domain, The amino acid sequence set forth in SEQ ID NO: 30 has at least 90% identity; or c) the light chain variable domain of a) and the heavy chain variable domain of b). The ST2 antibody of claim 1, wherein the light chain variable domain is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:96. The ST2 antibody of claim 1 or 2, wherein the heavy chain variable domain is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:30. An isolated ST2 antibody comprising: a) a light chain variable domain that differs from the amino acid sequence set forth in SEQ ID NO: 96 by no more than 10 amino acid additions, deletions or substitutions; b) and SEQ ID NO: 30 wherein the amino acid sequence differs by no more than 10 amino acid addition, deletion or substitution of the heavy chain variable domain; or c) a) of the light chain variable domain and b) Chain variable domain. The ST2 antibody of claim 4, wherein the light chain variable domain differs from the amino acid sequence set forth in SEQ ID NO: 96 by no more than 5 amino acid additions, deletions or substitutions. The ST2 antibody of claim 4 or 5, wherein the heavy chain variable domain differs from the amino acid sequence set forth in SEQ ID NO: 30 by no more than 5 amino acid additions, deletions or substitutions. The ST2 antibody of any one of claims 1, 2, 4, and 5, wherein the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO:96. The ST2 antibody of any one of claims 1, 2, 4, and 5, wherein the heavy chain variable structure The domain comprises the amino acid sequence set forth in SEQ ID NO:30. An isolated ST2 antibody comprising: a) a light chain variable domain comprising: LCDR1 differing from the LCDR1 sequence described in SEQ ID NO: 107 by no more than 3 amino acid additions, deletions or substitutions; The LCDR2 sequence described in ID NO: 118 differs by no more than 3 amino acid additions, deletions or substitutions of LCDR2; and differs from the LCDR3 sequence described in SEQ ID NO: 129 by no more than 3 amino acid additions, deletions or substitutions LCDR3; and b) a heavy chain variable domain comprising: HCDR1 differing from the HCDR1 sequence set forth in SEQ ID NO: 41 by no more than 3 amino acid additions, deletions or substitutions; and SEQ ID NO: 52 The HCDR2 sequence differs by no more than 3 amino acid additions, deletions or substitutions of HCDR2; and HCDR3 differs from the HCDR3 sequence set forth in SEQ ID NO: 63 by no more than 3 amino acid additions, deletions or substitutions. The isolated ST2 antibody of any one of claims 1, 2, 4, 5, and 9, wherein the light chain variable region comprises D28 or a conservative substitution thereof, I29 or a conservative substitution thereof, S30 or a conservative substitution thereof, N31 or Its conservative substitution, Y32 or its conservative substitution, Y49 or its conservative substitution, D50 or its conservative substitution, N53 or its conservative substitution, E55 or its conservative substitution, T56 or its conservative substitution, D91 or its conservative substitution, D92 or its conservation Substitution, N93 or a conservative substitution thereof, F94 or a conservative substitution thereof or L96 or a conservative substitution thereof. The ST2 antibody of claim 10, wherein the light chain variable region comprises D28 or a conservative substitution thereof, N31 or a conservative substitution thereof, D50 or a conservative substitution thereof, N53 or a conservative substitution thereof, E55 or a conservative substitution thereof, D91 or Its conservative substitution and D92 or its conservative substitution. The ST2 antibody of claim 11, wherein the light chain variable region comprises D28, N31, D50, N53, E55, D91 and D92. The isolated ST2 antibody of any one of claims 1, 2, 4, 5, and 9, wherein the heavy chain variable region comprises W33 or a conservative substitution thereof, I50 or a conservative substitution thereof, D57 or its Suffix substitution, R59 or its conservative substitution, H99 or its conservative substitution, G100 or its conservative substitution, T101 or its conservative substitution, S102 or its conservative substitution, S103 or its conservative substitution, D104 or its conservative substitution, Y105 or its conservative substitution Or Y106 or its conservative substitution. The ST2 antibody of claim 13, wherein the heavy chain variable region comprises S102 or a conservative substitution thereof, S103 or a conservative substitution thereof, D104 or a conservative substitution thereof, and Y105 or a conservative substitution thereof. The ST2 antibody of claim 14, wherein the heavy chain variable region comprises S102, S103, D104 and Y105. The ST2 antibody of any one of claims 1, 2, 4, 5, and 9, wherein the light chain variable domain comprises an LCDR1 as described in SEQ ID NO: 107; as described in SEQ ID NO: 118 An LCDR2 sequence and an LCDR3 sequence as set forth in SEQ ID NO: 129; and the heavy chain variable domain comprises the HCDR1 set forth in SEQ ID NO: 41; the HCDR2 sequence set forth in SEQ ID NO: 52 And the HCDR3 sequence as set forth in SEQ ID NO:63. The ST2 antibody of any one of claims 1, 2, 4, 5, and 9, wherein the antibody specifically binds human ST2 with an affinity of less than or equal to 1 x 10 ^-10 M. The ST2 antibody of any one of claims 1, 2, 4, 5, and 9, wherein the antibody inhibits human ST2 binding to human IL-33. The ST2 antibody of any one of claims 1, 2, 4, 5, and 9, wherein the antibody reduces ST2 signaling mediated by human IL-33 in human ST2 expressing cells. The ST2 antibody of claim 18, wherein the antibody inhibits cynomolgus monkey ST2 binding to cynomolgus IL-33. The ST2 antibody of claim 20, wherein the antibody reduces IL-33 mediated cynomolgus ST2 signaling in cynomolgus ST2 expressing cells. The ST2 antibody of any one of claims 1, 2, 4, 5, and 9, wherein the anti-systematic person Class antibodies. The ST2 antibody of claim 22, which comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence set forth in SEQ ID: 85 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 19. An isolated nucleic acid encoding a light chain variable domain of an antibody of any one of claims 1 to 23. An isolated nucleic acid encoding a heavy chain variable domain of an antibody according to any one of claims 1 to 23. An isolated nucleic acid encoding a light chain variable domain and a heavy chain variable domain of an antibody of any one of claims 1 to 23. The isolated nucleic acid of claim 24, wherein the nucleic acid encodes an antibody light chain. The isolated nucleic acid of claim 27, wherein the light chain is encoded by a nucleic acid comprising a nucleotide sequence that is at least 80% identical to the nucleotide sequence set forth in SEQ ID NO:74. The isolated nucleic acid of claim 27, wherein the light chain is encoded by a nucleic acid comprising a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:74. The isolated nucleic acid of claim 27, wherein the light chain is encoded by a nucleic acid comprising a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO:74. The isolated nucleic acid of claim 27, wherein the light chain is encoded by a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:74. The isolated nucleic acid of claim 25, wherein the nucleic acid encodes an antibody heavy chain. The isolated nucleic acid of claim 32, wherein the heavy chain is encoded by a nucleic acid comprising a nucleotide sequence that is at least 80% identical to the nucleotide sequence set forth in SEQ ID NO: 8. The isolated nucleic acid of claim 32, wherein the heavy chain is comprised of SEQ ID NO: 8 A nucleic acid encoding a nucleotide sequence in which the nucleotide sequence is at least 90% identical. The isolated nucleic acid of claim 32, wherein the heavy chain is encoded by a nucleic acid comprising a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 8. The isolated nucleic acid of claim 32, wherein the heavy chain is encoded by a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 8. A performance vector comprising the isolated nucleic acid of any one of claims 24 to 36. A recombinant host cell comprising the isolated nucleic acid of any one of claims 24 to 36 operably linked to a promoter. A recombinant host cell comprising the expression vector of claim 37. The recombinant host cell of claim 39, wherein the host cell secretes an antibody that binds to ST2. The recombinant host cell of any one of claims 39 to 40, wherein the cell line is derived from a mammalian cell. The recombinant host cell of claim 41, wherein the cell line is a Chinese hamster ovary (CHO) cell line. Use of an ST2 antibody according to any one of claims 1 to 23 for the manufacture of a medicament for the treatment of an autoimmune or inflammatory condition. The use of claim 43, wherein the antibody comprises a light chain variable domain amino acid sequence as set forth in SEQ ID NO: 96 and a heavy chain variable domain amine group as set forth in SEQ ID NO: Acid sequence. The use of claim 44, wherein the antibody comprises the light chain amino acid sequence as set forth in SEQ ID NO: 85 and the heavy chain amino acid sequence as set forth in SEQ ID NO: 19. The use of any one of claims 43 to 45, wherein the antigen binding protein inhibits IL-33 binding to ST2. The use of claim 46, wherein the autoimmune or inflammatory condition is asthma, different Position dermatitis, chronic obstructive pulmonary disease, pulmonary fibrosis, sepsis and trauma, systemic lupus erythematosus, inflammatory bowel disease, rheumatoid arthritis, sclerosis, Wegener's granulomatosis, Behchet Disease), cardiovascular disease, sinusitis, nasal polyps or eosinophilic bronchitis. A method of producing an ST2 antibody, the method comprising: a) cultivating the recombinant host cell of any one of claims 39 to 42; and b) isolating the ST2 antibody from the culture. An isolated ST2 antibody, wherein the ST2 antibody is cross-competing with an antibody comprising a light chain comprising the amino acid sequence set forth in SEQ ID: 85 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19. The ST2 antibody is isolated according to claim 49, wherein the ST2 antibody system is the ST2 antibody of any one of claims 1 to 23. An isolated ST2 antibody, wherein the ST2 antibody and the amino acid comprising amino acids 19 to 322 of SEQ ID NO: 1 are in the amino acid of SEQ ID NO: 1 from 33 to 44, as determined by hydrogen/oxime exchange analysis. Or combine within 88 to 94. The ST2 antibody is isolated as claimed in claim 51, wherein the ST2 antibody system binds within amino acids 33 to 44 and 88 to 94 of SEQ ID NO: 1 as determined by hydrogen/oxime exchange analysis. The ST2 antibody is isolated according to claim 52, wherein the ST2 binding protein is the ST2 binding protein of any one of claims 1 to 23. An isolated ST2 antibody that binds to ST2 to create an interface, wherein the interface produced by the binding comprises ST2 residues K1, F2, P19, R20, Q21, G22, K23, Y26, I70, V71, R72, S73 , P74, T75, F76, N77, R78, T79 or Y81. The ST2 antibody of claim 54 is isolated, wherein the interface produced by the binding comprises the ST2 residue P19, R20, Q21, G22, K23 or Y26. The ST2 antibody of claim 55, wherein the interface produced by the binding comprises ST2 residues P19, R20, Q21, G22, K23 and Y26. The ST2 antibody of claim 54, wherein the interface produced by the binding comprises ST2 residues I70, V71, R72, S73, P74, T75, F76, N77, R78, T79 or Y81. The ST2 antibody of claim 57, wherein the interface produced by the binding comprises ST2 residues I70, V71, R72, S73, P74, T75, F76, N77, R78, T79 and Y81. The ST2 antibody of claim 54, wherein the interface produced by the binding comprises ST2 residues P19, R20, Q21, G22, K23, Y26, I70, V71, R72, S73, P74, T75, F76, N77 , R78, T79 and Y81. The ST2 antibody of claim 59, wherein the interface produced by the binding comprises ST2 residues K1, F2, P19, R20, Q21, G22, K23, Y26, I70, V71, R72, S73, P74, T75 , F76, N77, R78, T79 and Y81. The isolated ST2 antibody of any one of claims 54 to 60, wherein the interface is determined by a difference in solvent exposure between the bound ST2 and the unbound ST2, wherein the interfacial residues are defined as having a difference greater than 10% Amino acids and their formation of water-mediated hydrogen bonds with the antibody. The isolated ST2 antibody of any one of claims 54 to 60, wherein the interface residues are defined as having at least one of the antibodies at Amino acid in the atom.